Recombinant human alpha-fetoprotein as an immunosuppressive agent

ABSTRACT

Disclosed are methods of inhibiting autoreactive immune cell proliferation in a mammal, involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an immune cell anti-proliferative fragment or analog thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/186,723 filed on Nov. 5, 1998, the disclosure ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to methods for treating autoimmunediseases.

[0003] Responses of the immune system initiate the destruction andelimination of invading organisms and toxic molecules produced by them.Because these immune reactions are destructive, it is essential thatthey be made in response only to molecules that are foreign to the hostand not to those of the host itself. The ability to distinguish foreignmolecules from self molecules is a fundamental feature of the immunesystem. Occasionally the immune system fails to make this distinctionand reacts destructively against the host's own molecules; suchautoimmune diseases can be fatal. Thus, tolerance to self antigensbreaks down, causing the components of the immune system such as T or Bcells (or both) to react against their own tissue antigens. Multiplesclerosis, rheumatoid arthritis, myasthenia gravis, insulin-dependentdiabetes mellitus, and systemic lupus erythematosus are a few examplesof such autoimmune diseases.

SUMMARY OF THE INVENTION

[0004] I have discovered that recombinant human alpha-fetoprotein madein a prokaryote (e.g., E. coli or baculovirus) or eukaryote is usefulfor inhibiting autoreactive immune cells derived from a mammal.Accordingly, the invention features a method of inhibiting transplantrejection in a mammal (e.g., a human patient), involving administeringto the mammal a therapeutically effective amount of recombinant humanalpha-fetoprotein or an immune cell anti-proliferative fragment oranalog thereof. Preferably, such immune cells include T cells or Bcells; and the recombinant human alpha-fetoprotein used in such methodsis produced in a prokaryotic cell (e.g., E. coli or baculovirus) oreukaryotic (e.g., transgenic animal) and is glycosylated orunglycosylated.

[0005] In another aspect, the invention features a method of inhibitinggraft-versus-host disease in a mammal (e.g., a human patient), involvingadministering to the mammal a therapeutically effective amount ofrecombinant human alpha-fetoprotein or an immune cell anti-proliferativefragment or analog thereof. Preferably, the recombinant humanalpha-fetoprotein used in such methods is produced in a prokaryotic cell(e.g., E. coli or baculovirus) or eukaryotic (e.g., transgenic animal)and is glycosylated or unglycosylated.

[0006] In yet another aspect, the invention features a method ofmitigating the side effects in a mammal (e.g. a human patient)undergoing chemotherapy, involving administering to the mammal atherapeutically effective amount of recombinant human alpha-fetoproteinor an immune cell anti-proliferative fragment or analog thereof.Preferably, the recombinant human alpha-fetoprotein used in such methodsis produced in a prokaryotic cell (e.g., E. coli or baculovirus) oreukaryotic (e.g., transgenic animal) and is glycosylated orunglycosylated.

[0007] In an additional aspect, the invention features a method ofmitigating the side effects in a mammal (e.g., a human patient)undergoing irradiation therapy, involving administering to the mammal atherapeutically effective amount of recombinant human alpha-fetoproteinor an immune cell anti-proliferative fragment or analog thereof.Preferably, the recombinant human alpha-fetoprotein used in such methodsis produced in a prokaryotic cell (e.g., E. coli or baculovirus) oreukaryote (e.g., transgenic animal) and is glycosylated orunglycosylated. In other preferred embodiments, such methods furtherinvolve administering to the mammal an immunosuppressive agent in aneffective dose that is lower than the standard dose when theimmunosuppressive agent is used by itself. Preferably, such animmunosuppressive agent is cyclosporine; is a steroid; is azathioprine;is FK-506; or is 15-deoxyspergualin. In yet another preferredembodiment, such a method involves administering to the mammal atolerizing agent. Preferably, the recombinant human alpha-fetoproteinused in such methods is produced in a prokaryotic cell (e.g., E. coli orbaculovirus) or eukaryote (e.g., transgenic animal) and is glycosylatedor unglycosylated.

[0008] By “immune cell anti-proliferative” is meant capable ofinhibiting the growth of an undesirable immune cell (e.g., anautoreactive T cell as measured using the assays described herein).

[0009] By “therapeutically effective amount” is meant a dose ofunglycosylated recombinant human alpha-fetoprotein (or a fragment oranalog thereof) capable of inhibiting autoreactive immune cellproliferation.

[0010] By “recombinant human alpha-fetoprotein” is meant a polypeptidehaving substantially the same amino acid sequence as the protein encodedby the human alpha-fetoprotein gene as described by Morinaga et al.,Proc. Natl. Acad. Sci., USA 80: 4604 (1983). The method of producingrecombinant human alpha-fetoprotein in a prokaryotic cell is describedin U.S. patent application Ser. No. 08/133,773 issuing as U.S. Pat. No.5,384,250.

[0011] According to the invention, administration of recombinant humanalpha-fetoprotein (“rHuAFP”) (or a fragment or analog thereof) can be aneffective means of preventing or treating or ameliorating autoimmunediseases in a mammal. To illustrate this, I have shown that recombinantHuAFP produced in a prokaryotic expression system is effective insuppressing T cell proliferation in response to self antigens, despitethe fact that such rHuAFP is not modified in the same fashion asnaturally occurring HuAFP. The use of natural HuAFP has heretofore beenlimited by its unavailability; natural HuAFP is obtained by laboriouspurification from limited supplies of umbilical cords and umbilical cordserum. Because biologically rHuAFP can now be prepared in largequantities using the techniques of recombinant DNA, the use of rHuAFPfor treating autoimmune diseases is now possible. The use of rHuAFP isespecially advantageous since there are no known adverse side effectsrelated to human alpha-fetoprotein and it is believed that relativelyhigh doses can be safely administered.

[0012] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0013] The drawings will first be described.

[0014] Drawings

[0015]FIG. 1A is an autoradiography showing the purity of recombinantAFP produced in E. coli. (ErAFP) on a 10% non-denaturing alkalinepolyacrylamide gel. Mouse amniotic fluid proteins (transferrin, AFP andalbumin) are shown in lane 1, natural human AFP (HuAFP) and ErAFP areshown in lane 2 and lane 3, respectively.

[0016]FIG. 1B is an autoradiography showing the purity of ErAFP producedin E. coli.on a 10% sodium dodecyl sulfate-polyacrylamide gel. Molecularweight markers are shown in lane 1, HuAFP and ErAFP are shown in lane 2and lane 3, respectively.

[0017]FIG. 1C is series of FPLC chromatograms showing the elutionprofile of HuAFP and ErAFP from a MonoQ anion exchange column. Thesuperimposed chromatograms identify HuAFP (Chromatogram 1) and ErAFP(Chromatogram 2).

[0018]FIG. 1D is a series of HPLC chromatograms showing the elutionprofile of HuAFP and ErAFP obtained by passing 50 μg of HuAFP and ErAFPthrough a reverse phase Delta Pak C18 column (Waters) and eluting with agradient of 0-100% acetonitrile in 0.1% TFA. The superimposedchromatograms identify HuAFP (Chromatogram 1) and ErAFP (Chromatogram2).

[0019]FIG. 2A is an autoradiograph showing the purity of recombinant AFPproduced in baculovirus (BrAFP) on a 10% non-denaturing alkalinepolyacrylamide gel. Protein samples are HuAFP (lane 2), BrAFP (lane 3),and ErAFP (lane 4). Molecular weight markers and mouse amniotic fluidare shown in lane 1.

[0020]FIG. 2B is an autoradiograph showing the purity of BrAFP on 10%SDS-acrylamide gel. Protein samples are HuAFP (lane 2), BrAFP (lane 3),and ErAFP (lane 4). Molecular weight markers and mouse amniotic fluidare shown in lane 1.

[0021]FIG. 2C is a series of FPLC chromatograms showing the elutionprofile of HuAFP, BrAFP, and ErAFP from a MonoQ anion exchange column.The superimposed chromatograms identify HuAFP (Chromatogram 1), BrAFP(Chromatogram 2), and ErAFP (Chromatogram 3).

[0022]FIG. 2D is a a series of HPLC chromatograms showing the elutionprofile of HuAFP, BrAFP, and ErAFP obtained by passing 50 μg of HuAFP,BrAFP, and ErAFP through a reverse phase Delta Pak C 18 column (Waters)and eluting with a gradient of 0-100% acetonitrile in 0.1% TFA. Thesuperimposed chromatograms identify natural HuAFP (Chromatogram 1),BrAFP (Chromatogram 2), and ErAFP (Chromatogram 3).

[0023]FIG. 3A is a graph showing the inhibitory effect of the ErAFP onthe kinetics of T cell activation. The proliferative responses weremeasured over a 4 day time course of cells cultured in the absence (∇)and in the presence of 100 μg/ml (▾) ErAFP. () denotes the backgroundproliferation of the responder cell population cultured separately.ErAFP-mediated suppression on the AMLR over the time course wassignificant (p<0.0 1).

[0024]FIG. 3B is a graph showing the dose-response relationship of ErAFPon autoproliferating T cells. The inhibition of autoproliferating Tcells was determined at 144 hours with amounts of ErAFP ranging from6-100 μg/ml (▾). (∇) denotes the control response of the reaction in theabsence of protein. Inhibition of autoreactive T cells by ErAFP in therange of 12.5-100 μg/ml is significant (p<0.005).

[0025]FIG. 4 is a bar graph showing that monoclonal anti-HuAFPantibodies (aAFP) block immunosuppression of the autologous mixedlymphocyte reactions (AMLR) by ErAFP (E. coli AFP). Immunosuppression byErAFP was significant (p<0.002) and blocking of ErAFP-mediatedimmunosuppression by monoclonal anti-HuAFP antibodies was alsosignificant (p<0.03).

[0026]FIG. 5 is a chart showing that monoclonal antibodies thatrecognize HuAFP block immunosuppression of AMLR by BrAFP and ErAFP.

[0027]FIG. 6 is a chart showing the immunosuppressive effects of BrAFP,ErAFP, and the AFP fragment of amino acids 1-22 (Δ(1-266)) on mitogenstimulated peripheral blood lymphocytes.

[0028]FIG. 7A is a bar graph showing the immunosuppressive effect ofhuman derived full-length HuAFP (HuAFP) versus human domain III AFP(HuDomIII) in AMLR. The gel insert confirms the size of the variousrecombinant AFP used in the AMLR assays: molecular weight markers (MW),1 μg HuAFP (lane 1), and 1 μg HuDom III (lane 2).

[0029]FIG. 7B is a graph showing the time course of HuAFP and HuDom IIIon immunosuppression of AMLR.

[0030]FIG. 8 is a schematic showing the nucleotide sequence (SEQ IDNO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the cDNAencoding human alpha-fetoprotein.

[0031]FIG. 9 is an autoradiograph showing the sizes of various AFP andAFP fragments (Lane A, MW marker; Lane B, HuAFP, Lane C, unpurifiedHuAFP and HuAFP Fragment I, Lane D, purified HuAFP Fragment I, and LaneE, purified full-length HuAFP).

[0032]FIG. 10A is a series of histograms showing an increased percentageof bone marrow cells (BM) that express the major histocompatibilty classI protein (MHC I) in presence of rHuAFP.

[0033]FIG. 10B is a bar graph showing an increased number of BM cellsthat express MHC I in the presence of rHuAFP.

[0034]FIG. 11A is a series of histograms showing an increased percentageof BM cells that express the major histocompatibilty class II protein(MHC II) in presence of rHuAFP.

[0035]FIG. 11B is a bar graph showing an increased number of BM cellsthat express MHC II in the presence of rHuAFP.

[0036]FIG. 12A is a series of FACS histogram patterns showing increasedexpression of the MHCI protein H-2K^(K) and increased expression of theMHCII protein I-2A^(k) in BM cells cultured in the presence of rHuAFP.

[0037]FIG. 12B is a series of bar graphs showing an increase in thepercent of BM cells that express H-2K^(K+) and I-2A^(K+) in eithermedium alone (control), rHuAFP, human albumin (HuAlb), or mouse albumin(MoAlb).

[0038]FIG. 13 is a bar graph showing the viability of BM cell culturestreated with 1% FCS in the presence or absence of IL-3, IL-7, or rHuAFP.

[0039]FIG. 14A is a bar graph showing the density of non-irradiated BMcells cultured in the presence of rHuAFP or Il-7.

[0040]FIG. 14B is a bar graph showing the density of irradiated BM cellscultured in the presence of rHuAFP or Il-7.

[0041]FIG. 15A is a series of bar graphs showing enhanced expression ofMHCI and enhanced density of MCHI-expressing BM in the presences ofrHuAFP after irradiation.

[0042]FIG. 15B is a series of bar graphs showing enhanced expression ofMHCII and enhanced density of MCHII-expressing BM in the presences ofrHuAFP after irradiation.

PRODUCTION OF RECOMBINANT HUMAN ALPHA-FETOPROTEIN

[0043] As summarized above, the invention includes therapies for theprevention and treatment of autoimmune diseases involving recombinanthuman alpha-fetoprotein (“rHuAFP”) or fragments or analogs thereof.

[0044] Production of Recombinant E. coli Alpha-Fetoprotein

[0045] Methods for producing such rHuAFP in a prokaryotic cell aredescribed in U.S. patent application Ser. No. 08/133,773 and in U.S.Pat. No. 5,384,250, issued Jan. 24, 1995, hereby incorporated byreference.

[0046] Expression, Purification, and Characterization of Recombinant AFPProduced in Baculovirus (BrAFP)

[0047] One eukaryotic expression system that is widely employed for theoverexpression of heterologous genes is the baculovirus expressionsystem. There are several advantages to generating recombinant proteinin baculovirus infected insect cells, including the ability of thisexpression system to produce high levels of soluble, secreted, andpost-translationally modified proteins (see, O'Reilly, et al.Baculovirus Expression Vectors. A Laboratory Manual. W. H. Freeman andCompany, N.Y., 1980). To investigate whether post-translationalmodifications play a role in mediating AFP immunomodulation, we comparedthe biological activities of a eukaryotic protein with that produced bya prokaryotic organism. E. coli was selected as the prokaryotic host inwhich to express AFP because it offered advantages such as ease andsimplicity in cloning and expressing a heterologous gene (see, Balbas &Bolivar. Gene Expression Technology, Goeddel, D. V. ed. New YorkAcademic Press, 1990), simple fusion protein strategies which ensuresgood translation initiation that may also permit one to overcome theinstability problems that can be encountered with small peptides, andrapid generation of biomass due to high rates of cell growth (Id.).

[0048] Western blot analysis of serum free supernatants from recombinantbaculovirus infected Sf9 cells detected a single immunoreactive bandwith monospecific anti-HuAFP Ab that was absent from the supernatant ofuninfected or wild-type virus-infected Sf9 cells. Passage of thesupernatant containing secreted BrAFP over ConA lectin chromatographyresulted in the binding of the recombinant protein and elution in theflow through of more than 90% of the contaminating proteins. Methyl α-Dmannopyranoside was used to elute BrAFP from the lectin column. Finalpurification of the BrAFP preparation was achieved by Mono Q FPLCchromatography, yielding a single polypeptide with an apparent molecularmass of 67 kD (FIG. 2B, lane 3). The BrAFP molecular weight is similarto that observed for the natural human molecule (FIG. 2B, lane 2). Thisresult, in addition to the binding of BrAFP to the ConA column,indicated that BrAFP was post-translationally modified viaglycosylation. However, the pattern of glycosylation of the BrAFP isexpected to differ from that of the native molecule, since Sf9 cellsinfected with recombinant baculovirus have been reported to be deficientin their ability to carry out complex glycosylation normally observedwith higher eukaryotic derived proteins (O'Reilly, supra; James, et al.Biotechnology 13:592-596, 1995). Purity of the isolated BrAFP wasverified by APAGE and SDS-PAGE (FIGS. 2A & 2B, lane 2, respectively),and is illustrated by a single symmetrical peal on FPLC and HPLCchromatograms as shown in FIG. 2C, graph 2, and FIG. 2D, graph 2,respectively. N-terminal analysis revealed that the melittin signalpeptide was cleaved from the mature recombinant human AFP polypeptide aspredicted.

[0049] Recombinant AFP expressed in E. coli represented approximately10% of total cell protein as determined by densitometric analysis ofCoomassie blue stained SDS-PAGE gels. Alkaline washes of lysed E. colipellets removed major contaminating proteins resulting in a 4-foldenrichment of ErAFP. The recombinant protein was solubulized bydissolving the pellet in a buffer containing guanidine andβ-mercaptoethanol, and subsequently refolded by rapid dilution of thedenaturant and reducing agent. Monomer BrAFP and ErAFP was efficientlyseparated from micro aggregates by employing Q-Sparse chromatography.Pure ErAFP was subsequently recovered as a single homogenous peak byFPLC Mono-Q anion exchange chromatography. The final product migrated at65 kD on SDS-PAGE (FIG. 2B, lane 4). Rechromatographed samples of pureErAFP on FPLC and HPLC yielded a single peak as shown in FIG. 2C, graph3 and FIG. 2D, graph 3 respectively, confirming the purity of the BrAFPand ErAFP preparation.

[0050] Baculovirus transfer vector pVT-PlacZ was modified by replacingthe MCS with the oligonucleotide 5′-GATCTAGAATTCGGATCCGGT-3′ and itscomplementary fragment, containing EcoR I and BamH I restrictions sitesin the 5′ to 3′ direction. The rHuAFP cDNA fragment was isolated asabove, inserted into the vector at the EcoR I and BamH I restrictionsties and transformations were verified for the presence and correctorientation of the rHuAFP cDNA fragment under the control of thepolyhedrin promoter by using restriction enzyme analysis.

[0051] Four mg of transfer vector pVT-PLacZ/HuAFP and 1 mg of linearizedwild-type AcMNPV baculovirus DNA (Invitrogen, San Diego, Calif.) wereco-transfected in the presence of 50 ml of Lipofectin Reagent (Gibco)into Spodoptera frigiperda (Sf9) cells. After 4-6 days of incubation,the transfection mixture was screened by a β-galactosidase assay and DNAslot-blot hybridizations for recombinant viruses containing both theμ-galactosidase and rAFP cDNA's. Sf9 cells seeded at a density of 1×10⁶cells/ml in 500 ml spinner flasks were infected with BrAFP baculovirusin serum-free Grace medium at a multiplicity of infection of 5. Thesupernatant was harvested at 72 hours post-infection by pelleting theSf9 cells at 200 X g for 10 minutes and the resultant media wasconcentrated 10-20 fold by ultrafiltration with a YM30 Amicon filtermembrane (Amicon).

[0052] Concentrated Grace media containing baculovirus produced AFP wasdialyzed against PBS overnight and then applied to a ConA Lectin column(Pharmacia) where all rAFP was bound to the column. Recombinant AFP(BrAFP) was eluted with 0.4M methyl a-D mannopyranoside and thisfraction was further purified by recovering protein from a FPLCanion-exchange MonoQ column (Pharmacia) using a linear NaCl gradientfrom 0-100% 1 M NaCl in 20 mM phosphate buffer pH 8.0. Purified BrAFPprotein preparations were dialyzed against 1x PBS and stored at −20° C.

[0053] Expression of Alpha-fetoprotein in Eukaryotes

[0054] Recombinant alpha-fetoprotein can be expressed in transgenicanimals. Transgenic animals may be prepared using methods well known tothe skilled artisan. For example, to prepare transgenic rodents such asmice, methods such as those set forth by Hogan et al., eds.(Manipulating The Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]) may be employed.Additionally, other animals that are suitable for expression of analpha-fetoprotein transgene include goat, sheep, and cow.

[0055] Recombinant apha-fetoprotein transgene expression can be directedto the milk of the transgenic animal. The alpha-fetoprotein transgene isgenerated in association with a mammary promoter to direct expression ofthe protein to the animal's milk; is introduced into the pronucleus of amammalian zygote (usually by microinjection into one of the two nuclei(usually the male nucleus) in the pronucleus); and thereafter implantedinto a foster mother. A proportion of the animals produced by the fostermother will carry and express the introduced gene that has integratedinto a chromosome. Usually the integrated gene is passed on to offspringby conventional breeding thus allowing ready expansion of stock.Preferably the alpha-fetoprotein is simply harvested from the milk offemale transgenic animals. Procedures for directing expression of a geneto the animal's milk are found in the following publications: Simons etal. (1988), Bio/Technology 6:179-183; Wright et al. (1991)Bio/Technology 9:830-834; U.S. Pat. No. 4,873,191 and; U.S. Pat. No.5,322,775. Manipulation of mouse embryos is described in Hogan et al,“Manipulating the Mouse Embryo; A Laboratory Manual”, Cold Spring HarborLaboratory 1986.

[0056] Mammalian cells (for example, CHO, COS, and myeloma cells) can beused as host for the expression of alpha-fetoprotein cDNAs and fragmentsthereof to produce the corresponding proteins and peptides. Forexpression of constructs leading to direct expression of active COS orCHO cell expression systems are preferred. The alpha-fetoprotein cDNAscan be introduced to plasmids and allowed to integrate into chromosomalDNA especially for CHO cells or allowed to replicate to very high copynumber especially in COS cells. The plasmids generally require aselectable marker for maintenance in transfected hosts, an efficienteukaryotic promoter to allow a high level of transcription from thecDNAs, convenient restriction enzyme sites for cloning andpolyadenylation, and transcription termination signals for messagestability. Several such vectors have been described in the literature(Bebbington, C. et al, 1992, Bio/Technology, vol 10, p169-175, andWright, A., 1991, Methods, vol 2, p125-135) and there are commerciallyavailable vectors, (such as pRc/CMV, Invitrogen Corp.) which aresuitable.

[0057] Fragments and Analogs

[0058] The invention includes biologically active fragments of AFP fromrHuAFP. A biologically active fragment of rHuAFP is one that possessesat least one of the following activities: (a) directs a specificinteraction with a target cell, e.g., binds to a cell expressing areceptor which is recognized by rHuAFP (e.g., the membrane of anautoreactive immune cell); or (b) halts, reduces, or inhibits the growthof an autoreactive immune cell (e.g., binds to a cell surface receptorand imparts an anti-proliferative signal); or (c) blocks, inhibits, orprevents an immunopathologic antibody reaction. The ability of rHuAFPfragments or analogs to bind to a receptor which is recognized by rHuAFPcan be tested using any standard binding assay known in the art. Methodsfor assaying the biological activity or rHuAFP fragments and analogs arealso known in the art, e.g., those described herein. Accordingly, arHuAFP fragment, like the full-length rHuAFP molecule, can be usedinhibit autoreactive immune cell proliferation.

[0059] In general, fragments of rHuAFP are produced according to thetechniques of polypeptide expression and purification described in U.S.patent application Ser. No. 08/133,773 (U.S. Pat. No. 5,384,250). Forexample, suitable fragments of rHuAFP can be produced by transformationof a suitable host bacterial cell with part of a HuAFP-encoding cDNAfragment (e.g., the cDNA described above) in a suitable expressionvehicle. Alternatively, such fragments can be generated by standardtechniques of PCR and cloned into the expression vectors (supra).Accordingly, once a fragment of rHuAFP is expressed, it may be isolatedby various chromatographic and/or immunological methods known in theart. Lysis and fractionation of rHuAFP-containing cells prior toaffinity chromatography may be performed by standard methods. Onceisolated, the recombinant protein can, if desired, be further purified,e.g., by high performance liquid chromatography (see, e.g., Fisher,Laboratory Techniques In Biochemistry And Molecular Biology, Work andBurdon, eds., Elsevier, 1980).

[0060] A rHuAFP fragment may also be expressed as a fusion protein withmaltose binding protein produced in E. coli. Using the maltose bindingprotein fusion and purification system (New England Biolabs), the clonedhuman cDNA sequence can be inserted downstream and in frame of the geneencoding maltose binding protein (malE), and the malE fusion protein canthen be overexpressed. In the absence of convenient restriction sites inthe human cDNA sequence, PCR can be used to introduce restriction sitescompatible with the vector at the 5′ and 3′ end of the cDNA fragment tofacilitate insertion of the cDNA fragment into the vector. Followingexpression of the fusion protein, it can be purified by affinitychromatography. For example, the fusion protein can be purified byvirtue of the ability of the maltose binding protein portion of thefusion protein to bind to amylose immobilized on a column.

[0061] To facilitate protein purification, the pMalE plasmid contains afactor Xa cleavage site upstream of the site into which the cDNA isinserted into the vector. Thus, the fusion protein purified as describedabove can then be cleaved with factor Xa to separate the maltose bindingprotein from a fragment of the recombinant human cDNA gene product. Thecleavage products can be subjected to further chromatography to purifyrHuAFP from the maltose binding protein. Alternatively, a fragment ofrHuAFP may be expressed as a fusion protein containing a polyhistidinetag can be produced. Such an alpha-fetoprotein fusion protein may thenbe isolated by binding of the polyhistidine tag to an affinity columnhaving a nickel moiety which binds the polyhistidine region with highaffinity. The fusion protein may then be eluted by shifting the pHwithin the affinity column. The rHuAFP can be released from thepolyhistidine sequences present in the resultant fusion protein bycleavage of the fusion protein with specific proteases.

[0062] Recombinant HuAFP fragment expression products (e.g., produced byany of the prokaryotic systems described in U.S. patent application Ser.No. 08/133,773 (U.S. Pat. No. 5,384,250)) may be assayed byimmunological procedures, such as Western blot, immunoprecipitationanalysis of recombinant cell extracts, or immunofluorescence (using,e.g., the methods described in Ausubel et al., Current Protocols InMolecular Biology, Greene Publishing Associates and Wiley Interscience(John Wiley & Sons), N.Y., 1994).

[0063] Once a fragment of rHuAFP is expressed, it is isolated using themethods described supra. Once isolated, the fragment of rHuAFP can, ifdesired, be further purified by using the techniques described supra.Fragments can also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, ThePierce Chemical Co., Rockford, Ill.). The ability of a candidate rHuAFPfragment to exhibit a biological activity of alpha-fetoprotein isassessed by methods known to those skilled in the art (e.g., thosedescribed herein).

[0064] The purified recombinant gene product or fragment thereof canthen be used to raise polyclonal or monoclonal antibodies against thehuman recombinant alpha-fetoprotein using well-known methods (seeColigan et al., eds., Current Protocols in Immunology, 1992, GreenePublishing Associates and Wiley-Interscience). To generate monoclonalantibodies, a mouse can be immunized with the recombinant protein, andantibody-secreting B cells isolated and immortalized with anon-secretory myeloma cell fusion partner. Hybridomas are then screenedfor production of recombinant human alpha-fetoprotein (or a fragment oranalog thereof)-specific antibodies and cloned to obtain a homogenouscell population that produces monoclonal antibodies.

[0065] As used herein, the term “fragment,” as applied to a rHuAFPpolypeptide, is preferably at least 20 contiguous amino acids,preferably at least 50 contiguous amino acids, more preferably at least100 contiguous amino acids, and most preferably at least 200 to 400 ormore contiguous amino acids in length. Fragments of rHuAFP molecules canbe generated by methods known to those skilled in the art, e.g.,proteolytic cleavage or expression of recombinant peptides, or mayresult from normal protein processing (e.g., removal of amino acids fromnascent polypeptide that are not required for biological activity).

[0066] Recombinant HuAFP fragments of interest include, but are notlimited to, Domain I (amino acids 1 (Thr)-197 (Ser), see FIG. 4, SEQ IDNO: 3), Domain II (amino acids 198 (Ser)-389 (Ser), see FIG. 4, SEQ IDNO: 4), Domain III (amino acids 390 (Gln)-590 (Val), see FIG. 4, SEQ IDNO: 5), Domain I+II (amino acids I (Thr)-389 (Ser), see FIG. 4, SEQ IDNO: 6), Domain II+III (amino acids 198 (Ser)-590 (Val), see FIG. 4, SEQID NO: 7), and rHuAFP Fragment I (amino acids 266 (Met)-590 (Val), seeFIG. 4, SEQ ID NO: 8). Activity of a fragment is evaluatedexperimentally using conventional techniques and assays, e.g., theassays described herein.

[0067] The invention further includes analogs of full-length rHuAFP orfragments thereof. Analogs can differ from rHuAFP by amino acid sequencedifferences, or by modifications (e.g., post-translationalmodifications), which do not affect sequence, or by both. Analogs of theinvention will generally exhibit at least 80%, more preferably 85%, andmost preferably 90% or even 99% amino acid identity with all or part ofa rHuAFP amino acid sequence. Modifications (which do not normally alterprimary sequence) include in vivo, or in vitro chemical derivatizationof polypeptides, e.g., acetylation, or carboxylation; such modificationsmay occur during polypeptide synthesis or processing or followingtreatment with isolated modifying enzymes. Analogs can also differ fromthe naturally occurring rHuAFP by alterations in primary sequence, forexample, substitution of one amino acid for another with similarcharacteristics (e.g., valine for glycine, arginine for lysine, etc.) orby one or more non-conservative amino acid substitutions, deletions, orinsertions which do not abolish the polypeptide's biological activity.These include genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, 1989, or Ausubel et al., supra)). Also included arecyclized peptide molecules and analogs which contain residues other thanL-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids, e.g., β or γ amino acids, or L-amino acids withnon-natural side chains (see e.g., Noren et al., Science 244:182, 1989).Methods for site-specific incorporation of non-natural amino acids intothe protein backbone of proteins is described, e.g., in Ellman et al.,Science 255:197, 1992. Also included are chemically synthesizedpolypeptides or peptides with modified peptide bonds (e.g., non-peptidebonds as described in U.S. Pat. No. 4,897,445 and U.S. Pat. No.5,059,653) or modified side chains to obtain the desired pharmaceuticalproperties as described herein. Useful mutants and analogs areidentified using conventional methods, e.g., those described herein.

[0068] The cloning, expression, isolation and characterization ofexemplary rHuAFP fragments now follows.

[0069] These examples are provided to illustrate, not limit, theinvention.

EXPERIMENTAL

[0070] MATERIAL AND METHOD

[0071] Polymerase Chain Reaction (PCR) rHuAFP Fragments

[0072] Plasmid constructs encoding fragments of human alpha-fetoproteinwere prepared using polymerase chain reaction (PCR) techniques known tothose skilled in the art of molecular biology, using oligonucleotideprimers designed to amplify specific portions of the humanalpha-fetoprotein gene (see e.g., PCR Technology, H. A. Erlich, ed.,Stockton Press, N.Y., 1989; PCR Protocols: A Guide to Methods andApplications, M. A. Innis, David H. Gelfand, John J. Sninsky, and ThomasJ. White, eds., Academic Press, Inc., N.Y., 1990, and Ausubel et. al.,supra).

[0073] The following six rHuAFP fragments were prepared to evaluatetheir biological activity (e.g., according to the methods disclosedherein):

[0074] Domain I Amino acids 1 (Thr)-197 (Ser), (FIG. 4, SEQ ID NO: 3)

[0075] Domain II Amino acids 198 (Ser)-389 (Ser), (FIG. 4, SEQ ID NO: 4)

[0076] Domain III Amino acids 390 (Gln)-590 (Val), (FIG. 4, SEQ ID NO:5)

[0077] Domain I+II Amino acids 1 (Thr)-389 (Ser), (FIG. 4, SEQ ID NO: 6)

[0078] Domain II+III Amino acids 198 (Ser)-590 (Val), (FIG. 4, SEQ IDNO: 7)

[0079] rHuAFP Fragment I Amino acids 266 (Met)-590 (Val), (FIG. 4, SEQID NO: 8)

[0080] Amino acid sequences were deduced from those for humanalpha-fetoprotein (1 (Thr)-590 (Val); SEQ ID NO: 2) shown in FIG. 4.Fragments of rHuAFP designated Domain I, Domain II, Domain III, DomainI+II, Domain II+III and rHuAFP Fragment I were synthesized usingstandard PCR reaction conditions in 100 μL reactions containing 34 μLH₂O, 10 μL 10X reaction buffer, 20 μL 1 mM dNTP, 2 μL DNA template(HuAFP cloned in pI 18), appropriate 5′ and 3′ oligonucleotide primers(10 μL 10 pmol/μL 5′ primer, 10 μL 10 pmol/μL 3′ primer), 1 μL glycerol,10 μL DMSO, and 1 μL Pfu polymerase (Stratagene, LaJolla, Calif.).Primers used for PCR amplifications were:

[0081] DomI25 5′-AAAAAAGGTACCACACTGCATAGAAATGAA-3′

[0082] (SEQ ID NO: 9)

[0083] DomI3 5′-AAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3′

[0084] (SEQ ID NO: 10)

[0085] DomII5 5-′AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3′

[0086] (SEQ ID NO: 11)

[0087] DomII3 5′-AAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3′

[0088] (SEQ ID NO: 12)

[0089] DomIII5 5′-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3′

[0090] (SEQ ID NO: 13)

[0091] DomIII3 5′-AAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3′

[0092] (SEQ ID NO: 14)

[0093] 5′rHuAFP Fragment I

[0094] 5′-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3′

[0095] (SEQ ID NO: 15)

[0096] Accordingly, primer pairs DomI25 and DomI3, DomII5 and DomII3,DomII5 and DomIII3, 5′rHuAFP Fragment I and DomIII3, DomI25 and DomII3,and DomII5 and DomIII3 were used to isolate cDNA sequences of Domain I,Domain II, Domain III, rHuAFP Fragment I, Domain I+II, and DomainII+III, respectively, of rHuAFP. Annealing, extension, and denaturationtemperatures were 50° C., 72° C., and 94° C., respectively, for 30cycles. PCR products were purified according to standard methods.Purified PCR products encoding Domain I and Domain I+II were digestedindividually with KpnI and BamHI and cloned separately intoKpnI/BamHI-treated pTrp4. Purified PCR products encoding Domain II,Domain III, Domain II+III, and rHuAFP Fragment I were digestedindividually with Bsp 106I and BamHI and were cloned separately intoBsp106I/BamHI-treated pTrp4. Each plasmid construct was subsequentlytransformed into competent E. coli cells. Since the expression productwill begin with the amino acid sequence encoded by the translation startsignal methionine, it is expected that such signal will be removed, orin any event, not affect the bioactivity of the ultimate expressionproduct.

[0097] Autologous Mixed Lymphocyte Reactions (AMLR)

[0098] AMLR assays were performed as described below.

RESULTS

[0099] Expression and Purification

[0100]E. coli containing the expression plasmid encoding rHuAFP FragmentI was cultured and purified. FIG. 9 (lane D) shows the SDS-PAGE profileof the purified rHuAFP Fragment I. N-terminal amino acid sequenceanalysis showed that rHuAFP Fragment I possessed the amino acid sequenceSer₂₆₇-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr₂₇₅ (SEQ ID NO: 16) whichcorresponds to the expected N-terminal amino acid sequence of rHuAFPFragment I (see FIG. 8, SEQ ID NO: 2) where the initiating methionine iscleaved intracellularly.

[0101] Inhibition of the Autologous Mixed Lymphocyte Reaction, (AMLR)

[0102] The immunosuppressive activity of 100 μg/ml rHuAFP Fragment I wasassessed by its ability to suppress human autologous mixed lymphocytereactions (AMLR). As shown in Table I, rHuAFP Fragment I inhibited theproliferative response of autoreactive lymphocytes stimulated byautologous non-T cells at 144 hours. TABLE I Thymidine IncorporationReaction Setup (CPM) T Cells 7118 ± 964  AMLR 83103 ± 6480  AMLR +rHuAFP Fragment I 29692 ± 2963  (100 μg/ml)

[0103] Recombinant HuAFP As An Immunosuppressive Agent

[0104] Immunosuppressive attributes of rHuAFP (or a fragment or analogthereof) were evaluated by any standard assay for analysis ofimmunoregulatory activity in vivo or in vitro. As discussed infra, theart provides a number of animal systems for in vivo testing ofimmunosuppressive characteristics of rHuAFP (or a fragment or analogthereof) on an autoimmune disease, e.g., the nonobese diabetic (NOD)mouse. Furthermore, a wide variety of in vitro systems are alsoavailable for testing immunosuppressive aspects of rHuAFP e.g., one suchin vitro assay evaluates the inhibition of auto antigen-inducedproliferation of T Cells in an autologous mixed lymphocyte reaction(AMLR).

[0105] The following examples demonstrate that unglycosylated rHuAFPinhibits T cell autoproliferation in response to self antigens. Theseexamples are provided to illustrate, not limit, the invention.

EXPERIMENTAL

[0106] MATERIALS AND METHODS

[0107] Gel Electrophoresis, Immunoblotting and Purification

[0108] The purity and characterization of rHuAFP was evaluated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andnondenaturing alkaline PAGE (APAGE) according to standard methods. Gelswere subsequently analyzed either by staining with Coomassie brilliantblue or by transferring electrophoretically separated polypeptides toImmobilon PVDF membranes (Millipore, Mississauga, ON) for immunoblottinganalysis. Recombinant HuAFP-monospecific rabbit anti-natural HuAFPpolyclonal antibody complexes were identified byalkaline-phosphatase-conjugated goat anti-rabbit IgG and theimmunoreactive bands were detected with the BCIP/NBT color developmentsolution (Bio-Rad Laboratories, Mississauga, ON) according to themanufacturer's instructions.

[0109] Column chromatography was performed according to standardmethods.

[0110] Cell Preparation

[0111] Human peripheral blood mononuclear cells (PBMC) were isolatedfrom heparin-treated whole blood of normal adult donors by venipuncture.Blood was diluted 1:1 with PBS, layered on Ficoll-Hypaque (Sigma, St.Louis, Mo.) and centrifuged at 400 x g for 30 min at 25° C. Cells wereremoved from the interface, washed 3 times in PBS and examined undermicroscope for viability using the Trypan Blue exclusion method. Cellpreparations which were less than 95% viable were discarded. At thisstage, the cell preparations were ready to be cultured for the Con Amitogen assay. For the autologous mixed lymphocyte reaction (AMLR), PBMCwere fractionated into T cell and non T cell populations. Responder Tcells for the AMLR were prepared by passing 1.5×10⁸ PBMC over acommercial Ig-anti-Ig affinity column (Biotex Laboratories, Edmonton,AB), washing 3 times in PBS, and resuspending them in RPMI-1640 medium.The separation of non-T from T cell populations in PBMC was based on thecapacity of T cells to form E-rossettes with sheep red blood cells(SBRC) (Mendas, et al. J. Immunol. 111:860-867, 1973). One ml ofpelleted SRBC was treated with 1 U of neuraminidase (Sigma) for 1 hourat 37° C., washed 3 times, and resuspended in 50 ml of RPMI media toyield a 2% SRBC solution. This procedure enhanced cellular interactionsbetween T-cells and SRBC. PBMC (5-9×10⁷) were then incubated in a ratioof 1×10⁷ PBMC:2 ml SRBC solution: 2 ml heat inactivated FCS at 37° C.for 10 min. This was followed by 5 min of centrifugation at 200 x gprior to a second incubation for 60 min at 4° C. The cell mixtures werethen gently resuspended and the rossetted T cells separated from thenon-T cells by density centrifugation on Ficoll-Hypaque for 30 min at400 x g. The non-T cells were collected from the interface, washed threetimes and resuspended in RPMI-1640 media.

[0112] Autologous Mixed Lymphocyte Reactions (AMLR)

[0113] Isolation of human PBMC, their fractionation into non-T cellpopulations, and the AMLR, were performed according by the standardprocedure described above. Responder T cells were isolated by passing1.5×10⁸ PMBC over a commercial Ig-anti-Ig affinity column (BiotekLaboratories) and 2×10⁵ responder cells were subsequently cultured with2×10⁵ autologous ¹³⁷Cs-irradiated (2500 rads) non-T stimulator cellsfrom a single donor. The medium employed consisted of RPMI-1640supplemented with 20 mM HEPES (Gibco), 5×10⁻⁵ M 2-mercaptoethanol (BDH,Montreal, QC), 4 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco) and100 μg/ml streptomycin sulfate, with the addition of 10% fresh humanserum autologous to the responder T cell donor for the AMLR. Varyingconcentrations of purified rHuAFP, human serum albumin (HSA), anti-HuAFPmonoclonal antibodies clone #164 (125 μg/ml final concentration inculture) (Leinco Technologies, St. Louis, Mo.) were added at theinitiation of cultures. AMLR cultures were incubated for 4 to 7 days, at37° C. in 95% air and 5% CO₂. At the indicated intervals, DNA synthesiswas assayed by a 6 hour pulse with 1 μCi of ³H-thymidine (specificactivity 56 to 80 Ci/mmole, ICN). The cultures were harvested on amultiple sample harvester (Skatron, Sterling, Va.), and theincorporation of ³H-TdR was measured in a Packard 2500 TR liquidscintillation counter. Results are expressed as mean cpm± the standarderror of the mean of triplicate or quadruplicate cultures.

[0114] Mitogen-stimulated Lymphocyte Assays

[0115] Mitogen cultures consisted of 2.5×10⁵ PBMC stimulated with 1βg/ml of Con A (Pharmacia). The media employed consisted of RPMI-1640supplemented with 20 mM HEPES (Gibco), 5×10⁵ M 2-mercaptoethanol (BDH,Montreal, QC), 4 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco) and100 mg/ml streptomycin sulfate and 2 mg/ml of low endotoxin human serumalbumin (HSA) (ICN Biomedials Canada, Mississauga, ON). Purified rAFPfrom both recombinant sources were added to the cultures at aconcentration of 100 μg/ml. Mitogen reactions were cultured for 48 hoursat 37° C. in 95% air and 5% CO₂ and assayed for proliferative responsesas described for the AMLR.

[0116] RESULTS

[0117] Expression and Purification of Human Alpha-Fetoprotein

[0118] Purity of isolated rHuAFP expressed in E. coli was verified as asingle band on Coomassie stained APAGE and SDS-PAGE are shown in FIGS.1A-1B, respectively. Soluble monomeric rHuAFP derived from E. coli wasobtained by eluting a protein fraction containing rHuAFP employingQ-sepharose chromatography. Approximately 1 mg of pure rHuAFP per literof bacterial culture was recovered as a single homogeneous peak by FPLCMono-Q anion exchange with 220-230 mM NaCl and migrated at approximately65 kD on SDS-PAGE (FIG. 1B). Recombinant HuAFP exhibits a lowermolecular weight on SDS-PAGE than natural HuAFP, since prokaryoticexpression systems lack the enzymatic machinery required forglycosylation of proteins. Rechromatographed samples of pure rHuAFP onFPLC and HPLC yielded a single peak as shown in FIG. 1C and FIG. 1D,confirming the purity of the rHuAFP preparation. In addition, N-terminalsequencing data correspond to the expected amino acid sequence at theN-terminus of rHuAFP.

[0119] Inhibition of the AMLR

[0120] The immunosuppressive activity of rHuAFP was assessed by itsability to suppress human AMLR. As shown in FIG. 3A, rHuAFP inhibitedthe proliferative response of autoreactive lymphocytes stimulated byautologous non-T cells, throughout the 4 to 7 day time course measuringautoproliferation. Results from dose-response studies performed at thepeak of T cell autoproliferation, as shown in FIG. 3B, demonstrate thatthe addition of rHuAFP at the initiation of cultures suppressed the AMLRin a dose-dependent manner. Furthermore, parallel viability studiesestablished that the inhibitory activity of rHuAFP on human autoreactiveT cells was not due to non-specific cytotoxic effects.

[0121] To further substantiate that rHuAFP was the agent responsible forthe inhibition of autoproliferating T cells, blocking of rHuAFP-mediatedsuppression of the AMLR was performed using commercial murine anti-humanAFP monoclonal antibodies (MAb). As illustrated in FIG. 4, suppressionof proliferating autoreactive T cells by 100 μg/ml of rHuAFP wascompletely blocked by anti-HuAFP MAb. The addition of 100 μg/ml of HSAdid not diminish the AMLR response and the presence of MAb alone in thereaction culture was without any effect.

[0122] Recombinant polypeptides produced in prokaryotic expressionsystems are at risk for contamination with host cell lipopolysaccharide(LPS) during their isolation from bacteria. It has been demonstratedthat small amounts of LPS can antagonize the biological activities ofcytokines, thereby impairing the immune responsiveness of macrophages.Accordingly, the effect of endotoxin on various rHuAFP preparations wasevaluated by performing AMLR experiments with recombinant proteindepleted of endotoxin by passage over Detoxi-gel (Pierce) versus that ofrHuAFP which was untreated. Results of these experiments showed thatboth preparations had equivalent levels of immunosuppressive activity.

[0123] As shown in FIG. 3A and FIG. 3B, the results of this study alsodemonstrate that rHuAFP suppresses the proliferation of autoreaction Tcells with a potency equivalent to glycosylated rHuAFP by elicitinginhibitory effects on autoproliferating T cells throughout the in vitroreactions, with highly significant inhibition being achieved with rHuAFPconcentrations ranging from 5 μg/ml to 100 μg/ml.

[0124] Suppression of autoproliferating and mitogen responsivelymphocytes by BrAFP

[0125] In order to address in a definitive manner whether post-syntheticalterations play a role in mediating the immunosuppressive properties ofAFP, we assessed the ability of BrAFP and ErAFP, which is notpost-translationally modified, to suppress the proliferative response ofautoreactive T cells in the AMLR. In FIG. 5, 100 μg/ml of BrAFP andErAFP added at the initiation of the AMLR suppressed thelymphoproliferative response by 57% and 58%, respectively. Moreover,BrAFP and ErAFP anti-proliferative activity was blocked by the additionof anti-human AFP Mab. An equivalent amount of HSA augmented thereaction. The possibility that AFP might be causing a shift in thekinetics of the AMLR was eliminated, when rHuAFP, at a concentration of100 μg/ml, was shown to inhibit autoreactive T lymphocytes fromproliferating in response to autologous non-T cells throughout theautoproliferation stages of the time course from 96 to 168 hours (FIG.3A).

[0126] We next examined the effects of various concentrations of rAFP onDNA synthesis in autoproliferating T cells. A representative experiment(FIG. 3B) demonstrates a marked dose-dependent inhibition of³H-thymidine incorporation, with significant anti-proliferative effectsstill observed at 12 μg/ml. Viability studies established that theinhibitory activity of rAFP on human autoreactive T cells was not due tonon-specific cytotoxic effects.

[0127] We carried out experiments in serum-free media to control for thepossibility that exogenous serum factors may interact with thegenetically engineered protein and mediate the anti-proliferativeactivity of recombinant human AFP. As shown in FIG. 6, experiments 1 and2 demonstrate that the addition of 100 μg/ml of either BrAFP or ErAFP toin vitro cultures containing mitogen stimulated PBMC in RPMI mediasupplemented with 2 mg/ml HSA reduced lymphoproliferation by more than60%. The addition of 100 μg/ml HSA also reduced lymphoproliferation bymore than 60%. The addition of HSA at 100 μg/ml had no effect on the ConA assay. These results demonstrate that neither post-translationalmodifications nor exogenous serum factors mediate AFP immunosuppression.

[0128] Endotoxin does not influence AFP-mediated immunosuppression

[0129] Recombinant polypeptides produced in prokaryotic expressionsystems are at risk for contamination with host cell lipopolysaccharides(endotoxin) during their isolation from bacteria. It has beendemonstrated that small amounts of LPS can antagonize the biologicalactivities of cytokines, thereby impairing the immune responsiveness ofmacrophages (Bogdan, et al. J. Immunol. 151:301-331, 1993). We thereforeevaluated the effect of endotoxin on various ErAFP preparations byperforming AMLR experiments with recombinant protein that had beentreated to remove endogenous endotoxin by passage over Detoxi-gel(Pierce) versus that ErAFP which was not subjected to the affinityresin. As shown in FIG. 5, Exp 3, a five fold reduction in the amount ofendotoxin to levels that are below those that stimulate the release ofinterleukin 1 from human monocytes (Duff, et al. J. Immunol. Methods52:323-331, 1982) did not alter the immunosuppressive activity of therecombinant protein.

[0130] Immunosuppression by a 35 kD Fragment corresponding to anNH₂-Terminus-Deletion of Full-length FRAFP

[0131] An immunoblot analysis of whole bacterial cell extractscontaining ErAFP identified, in addition to the 67 kD whole AFPmolecule, an immunoreactive protein band with an approximate molecularweight of 35 kD. This protein was purified on MonoQ FPLC. Amino terminalsequencing of the 35 kD fragment revealed that this polypeptidecorresponded to the COOH two-thirds of full-length AFP, beginning atamino acid position 267: 1                5                    10Ser-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr-Leu-

[0132] Consequently, we wanted to determine whether this truncated AFPfragment termed AFP Δ(1-226) retained the immunosuppressive activitythat is observed with the intact molecule. For comparison, the 25 kDfragment was evaluated in parallel with the complete ErAFP molecule forits ability to down regulate in vitro T cell proliferative reactions. Itwas observed that the AFP Δ(1-226) polypeptide was similar tofull-length rAFP with respect to mediating immunoregulation, suppressingthe AMLR throughout the kinetics of autoproliferation (FIG. 3) andinhibiting mitogen induced PBL proliferation by 61% (FIG. 6, Exp. 3).This finding indicates that the first 266 amino acids of AFP are notrequired for immunoregulation.

[0133] Generation of a bioactive AFP fragment corresponding to Domain 3

[0134] The previous study indicated that immunoregulatory active sitesare present within the last two thirds of Domain 2 and intact Domain 3.Thus, a gene segment corresponding to the third domain of AFP (Morinaga,et al. Proc. Natl. Acad. Sci. USA 80:4604, 1983) was cloned by PCR intoE. coli. The protein was identified by immunoblot employing anti-humanAFP polyclonal antibodies and was subsequently purified by Q-sepharoseand Mono Q anion exchange chromatography. The inhibitory activity ofDomain 3 on autoproliferating and mitogen induced proliferating Tlymphocytes was performed in parallel with full-length rAFP. As shown inthe representative experiment in FIG. 7A, the truncated AFP segmentsuppressed Con A stimulated PBL's by 60% and inhibited the AMLR by 79%,whereas full-length rAFP downregulated the same in vitro responses by50% and 58% respectively. These results demonstrate that active sitesfor immunoregulation exist in the latter third of the AFP molecule.

[0135] Autoimmune Disease

[0136] As is discussed above, autoimmune diseases are characterized by aloss of tolerance to self antigens, causing cells of the immune systems,e.g., T or B cells (or both), to react against self tissue antigens.Autoimmune diseases may involve any organ system, although some areaffected more commonly than others. Examples of tissues affected byautoimmune conditions include: the white matter of the brain and spinalcord in multiple sclerosis; the lining of the joints in rheumatoidarthritis; and the insulin secreting β islet cells of the pancreas ininsulin-dependent diabetes mellitus. Other forms of autoimmune diseasedestroy the connections between nerve and muscle in myasthenia gravis ordestroy the kidneys and other organs in systemic lupus erythematosus.Examples of other autoimmune diseases include, without limitation,Addison's disease, Crohn's disease, Graves' disease, psoriasis,scleroderma, and ulcerative colitis.

[0137] The art provides a wide variety of experimental animal systems,transgenic and non-transgenic, for testing therapies for human illnessinvolving autoimmune diseases (see e.g., Paul, W. E., FundamentalImmunology, 2nd ed., Raven Press, N.Y., 1989; and Kandel et al.Principles of Neural Science, 3rd ed., Appleton and Lange, Norwalk,Conn., 1991; and Current Protocols In Immunology, Coligan, J. E.,Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, eds.,Green Publishing Associates (John Wiley & Sons), N.Y., 1992). Based onthe above-described experimental results showing immunosuppressiveactivity of unglycosylated rHuAFP, it is reasonable to believe thatother autoimmune diseases can be treated by administration of suchrHuAFP (or fragment or analog thereof) produced in a prokaryotic system.Accordingly, the invention provides the use of rHuAFP (or a fragment oranalog thereof) for treatment (i.e., prevention or suppression oramelioration or promotion of remission) of any autoimmune disease.

[0138] There now follow examples of animal systems useful for evaluatingthe efficacy of recombinant human alpha-fetoprotein or an immune cellanti-proliferative fragment or analog thereof in treating autoimmunediseases. These examples are provided for the purpose of illustrating,not limiting, the invention.

[0139] Multiple Sclerosis

[0140] Multiple sclerosis (MS) is a demyelinating disease involvingscattered areas of the white matter of the central nervous system. InMS, myelin basic protein and proteolipid protein are the major targetsof an autoimmune response involving T lymphocytes, among other immunesystem components. Loss of the myelin sheath of nerve cells(demyelination) occurs, resulting in neurological symptoms thatculminate in coma or paralysis.

[0141] Experimental autoimmune encephalomyelitis (EAE) is a primarymodel used in the art to examine and assess the effectiveness oftherapeutic agents for treating MS. EAE is an inflammatory autoimmunedemyelinating disease induced in laboratory animals by immunization withcentral nervous system tissue. When animals (e.g., mice, rats, guineapigs, rabbits, monkeys, etc.) are injected with adjuvant, e.g., completeFreund's adjuvant, plus myelin basic protein or proteolipid protein, EAEis induced, which is similar, pathologically to MS (see e.g., Alvord etal., Experimental Allergic Encephalomyelitis—A Useful Model for MultipleSclerosis, Liss, N.Y., 1984; Swanborg, Meth. Enzymol 162:413, 1988; andMcCarron et al., J. Immunol., 147: 3296, 1991.)

[0142] To evaluate rHuAFP or a fragment or analog thereof, EAE isinduced in an appropriate laboratory animal, e.g., a mouse or rabbit,according to methods known in the art. To evaluate the compound'simmunosuppressive effect on EAE, i.e., its ability to prevent orameliorate EAE, the compound is administered according to standardmethods, e.g., intravenously or intraperitoneal, at an appropriatedosage on a daily basis. Generally, administration is initiated prior toinducing EAE and/or after the clinical appearance of EAE. Controlanimals receive a placebo, e.g., human serum albumin, similarlyadministered as for rHuAFP or related molecules. The effect of the testmolecules on EAE is monitored according to any standard method. Forexample, weight loss and muscle paralysis in EAE-induced animals ismonitored on a daily basis. If desired, histological inspection (e.g.,by using any standard histochemical or immunohistochemical procedure,see e.g., Ausubel et al., Current Protocols In Molecular Biology, GreenePublishing Associates (John Wiley & Son), N.Y., 1994; Bancroft andStevens, Theory and Practice of Histochemical Techniques, ChurchillLivingstone, 1982) of brain and spinal cord tissues is performed andtissue samples examined microscopically for evidence of EAE, e.g.,evidence of perivascular cellular infiltrates. Comparative studiesbetween treated and control animals are used to determine the relativeefficacy of the test molecules in preventing or ameliorating EAE. Amolecule which prevents or ameliorates (decreases or suppresses orrelieves or promotes remission of) the symptoms of EAE is considereduseful in the invention.

[0143] Rheumatoid Arthritis

[0144] Rheumatoid arthritis (RA) is a common chronic illness in whichthe synovial membrane of multiple joints becomes inflamed, causingdamage to cartilage and bone. RA is associated with human lymphocyteantigen (HLA)-DR4 and considered to be an autoimmune disorder involvingT cells, see e.g., Sewell et al., Lancet 341: 283, 1993. RA results froma complex interaction of synovial cells with various cellular elements(and their soluble products) that infiltrate from the circulation intothe synovial lining of joints. A series of biological events occur whichultimately lead to a lesion which invades and erodes collagen and thecartilage matrix of the joint.

[0145] A number of animal models of RA, e.g., the MRL-lpr/lpr mouse, areknown in the art which develop a form of arthritis resembling the humandisease (see e.g., Fundamental Immunology, supra). Alternatively,autoimmune collagen arthritis (ACA) and adjuvant arthritis (AA) can beinduced in an appropriate animal according to standard methods.

[0146] To evaluate rHuAFP or a fragment or analog thereof onimmunosuppressive on RA, i.e., the compound's ability to prevent orameliorate RA, the test molecule is administered to a MRL-lpr/lpr mouseaccording to standard methods, e.g., intravenously or intraperitoneally,at an appropriate dosage on a daily basis. Generally, administration isinitiated prior to the onset of RA and/or after the clinical appearanceof RA. Control animals receive a placebo, e.g., human serum albumin,similarly administered as for rHuAFP or related molecules. The effect ofthe test molecule on RA is monitored according to standard methods. Forexample, analysis of the cellular component(s) of a synovial joint aremonitored on a daily basis. If desired, histological inspection (e.g.,by using any standard histochemical or immunohistochemical procedure,see e.g., Ausubel et al., supra; Bancroft and Stevens, supra) of thesynovial joint is performed and tissue samples examined microscopicallyfor evidence of RA, e.g., evidence of erosion of collagen and cartilagematrix in a joint. Comparative studies between treated and controlanimals are used to determine the relative efficacy of the test moleculein preventing or ameliorating RA. A test molecule which prevents orameliorates (decreases or suppresses or relieves or promotes remissionof) the symptoms of RA is considered useful in the invention.

[0147] Myasthenia Gravis

[0148] Myasthenia gravis (MG) is a disorder of neuromusculartransmission in which there are autoantibodies against acetylcholinereceptors of neuromuscular junctions. Antibodies attack the junction,causing weakness and paralysis. Females are afflicted twice as often asmales, typically during the third decade of life. Muscular weakness isthe predominant feature of the disease. Clinical signs include droopingof the eyelids and double vision. There is an association between MG andhyperthyroidism.

[0149] Experimental autoimmune MG (EAMG) has been studied in a varietyof animals including rabbits, monkeys, Lewis rats and inbred strains ofmice (see e.g., Principles of Neural Science, supra), the symptoms ofEAMG resemble the essential characteristics of the human disease. Asingle injection of acetylcholine receptor, e.g., purified from theelectric organs of the eel Torpedo californica, along with adjuvants,causes an acute phase of weakness within 8 to 12 days and then chronicweakness after about 30 days. The response to the eel receptor is T celldependent. The C57BL/6 strain (H-2^(B)) is a high responder to Torpedoreceptor and highly susceptible to EAMG.

[0150] To evaluate rHuAFP or a fragment or analog thereof, EAMG isinduced in an appropriate laboratory animal, e.g., the C57BL/6 strain(H-2^(B)) mouse, according to methods known in the art. To evaluate thecompound's immunosuppressive effect on EAMG, i.e., its ability toprevent or ameliorate EAMG, the compound is administered according tostandard methods, e.g., intravenously or intraperitoneally, at anappropriate dosage on a daily basis. Generally, administration isinitiated prior to inducing EAMG and/or after the clinical appearance ofEAMG. Control animals receive a placebo, e.g., human serum albumin,similarly administered as for rHuAFP or related molecules. The effect ofthe test molecules on EAMG is monitored according to standard methods.For example, nerve stimulation in an electromyographic muscle assay(e.g., according to the methods of Pachner et al., Ann. Neurol. 11:48,1982) in EAMG-induced animals can be assayed. If desired, histologicalinspection (e.g., by using any standard histochemical orimmunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroftand Stevens, supra) of tissue samples is performed and tissue samplesexamined microscopically for evidence of EAMG, e.g., evidence ofmonocyte infiltration and/or autoantibody localization at acetylcholinereceptors of neuromuscular junctions. Comparative studies betweentreated and control animals are used to determine the relative efficacyof the test molecules in preventing or ameliorating EAMG. A moleculewhich prevents or ameliorates (decreases or suppresses or relieves orpromotes remission of) the symptoms of EAMG is considered useful in theinvention.

[0151] Insulin-Dependent Diabetes Mellitus

[0152] Diabetes is a disorder of glucose metabolism. Insulin-dependentdiabetes mellitus (IDDM), also known as Type I diabetes, is anautoimmune disease characterized by T-cell mediated destruction ofpancreatic β cells in the islets of Langerhans, accompanied by an immuneresponse to a diversity of self peptides leading to hyperglycemia, amongother pathological events. IDDM patients depend on exogenous insulin tomaintain normal glucose metabolism. Humans at risk for developing IDDMcan be identified prior to onset of hyperglycemia by the abnormaloccurrence of autoantibodies to insulin, islet cells, glutamic acidcarboxylase, as well as other autologous proteins (see e.g., Baekkeskovet al., J. Clin. Invest. 79:926, 1987; Dean et al., Diabetologia 29:339, 1986; Rossini et al., Annu. Rev. Immunol. 3:289, 1985; Srikanta etal., N. Engl. J Med. 308:322, 1983). Autoantibody patterns, in general,are predictive for the eventual disease progression and/or risk fordeveloping the disease (see e.g., Keller et al., Lancet 341:927, 1993).

[0153] Examples of animal models which spontaneously develop IDDMresembling the human disease include the Bio-Breeding (BB) rat andnonobese diabetic (NOD) mouse. Diabetes is also experimentally inducedby streptozotocin.

[0154] The BB rat spontaneously develops a disease similar to IDDM, withinsulitis (infiltration of mononuclear cells into the pancreatic islets)and autoantibodies against self cells and insulin (see e.g., Baekkeskovet al., J Clin. Invest. 79:926, 1987; Rossini et al, supra; Nakhooda etal., Diabetes 26: 100, 1977; Dean et al., Clin. Exp. Immunol. 69: 308,1987).

[0155] NOD mice typically develop insulitis between 5 and 8 weeks ofage, and by 7 months 70% of the females and 40% of the males becomediabetic. T cells transferred from diabetic mice to young nondiabeticNOD mice induce diabetes within 2 to 3 weeks (see e.g., Bendelac et al.,J. Exp. Med. 166:823, 1987). NOD mice usually die within 1 to 2 monthsafter the onset of diabetes unless they receive insulin therapy.

[0156] Chemically induced diabetes is accomplished using multipleinjections of small doses of streptozotocin, a drug toxic for pancreaticβ cells, which causes severe insulitis and diabetes (see e.g., Kikutaniet al., Adv. Immunol. 51:285, 1992).

[0157] Accordingly, the art provides a variety animal models resemblinghuman IDDM which can be used to examine and assess approaches for theprevention or amelioration of diabetes involving rHuAFP (or a fragmentor analog thereof).

[0158] To evaluate the immunosuppressive effect of rHuAFP or a fragmentor analog thereof on the development of diabetes mouse, i.e., thecompound's ability to treat or prevent insulitis and diabetes, the testcompound is administered to an appropriate test animal, e.g, a NODmouse, according to standard methods, e.g., intravenously orintraperitoneally, at an appropriate dosage on a daily basis. Generally,administration is initiated prior to the onset of insulitis and diabetesand/or after the clinical appearance of diabetic characteristics.Control animals receive a placebo, e.g., human serum albumin, similarlyadministered as for rHuAFP or related molecules. The effect of testmolecules on insulitis and diabetes is monitored according to standardmethods. For example, weight loss, ketone body formation, and bloodglucose concentration is monitored on a daily basis. If desired,histological inspection (e.g., by using any standard histochemical orimmunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroftand Stevens, supra) of pancreatic islet cells is performed and tissuesamples examined microscopically for evidence of insulitis and β celldestruction. Comparative studies between treated and control animals areused to determine the relative efficacy of the test molecules inpreventing or ameliorating the diabetic condition. A molecule whichprevents or ameliorates (decreases or suppresses or relieves or promotesremission of) the symptoms of diabetes, e.g., IDDM, is considered usefulin the invention.

[0159] Systemic Lupus Erythematosus

[0160] Systemic lupus erythematosus (SLE) is a severe systemicautoimmune disease. About 90% of patients with this disease are youngwomen. This marked preponderance of females is not seen before pubertyor after menopause. The illness generally begins in young adulthood whena characteristic skin rash appears over cheekbones and forehead. Hairloss is common, as is severe kidney damage, arthritis, accumulation offluid around the heart and inflammation of the lining of the lungs. Innearly half of the patients the blood vessels of the brain also becomeinflamed, leading to paralysis and convulsions. The activity of thedisease, like other autoimmune diseases, can fluctuate: long quiescentperiods of good health can terminate abruptly and inexplicably with theonset of a new attack. A large number of different autoantibodies areknown to occur in SLE, e.g., autoantibodies against DNA, RNA andhistones (see, e.g., Fundamental Immunology, supra)

[0161] A number of animal models of human SLE, e.g., inbred mousestrains including NZB mice and their F₁ hybrids, MRL mice, and BXSBmice, are known in the art (see e.g., Bielschowsky et al. Proc. Univ.Otago Med. Sch. 37:9, 1959; Braverman et al., J. Invest. Derm. 50: 483,1968; Howie et al. Adv. Immunol. 9:215, 1968; Genetic Control ofAutoimmune Disease, Rose, M., Bigazzi, P. E., and Warner, N. L. eds.,Elsevier, Amsterdam, 1979; and Current Protocols In Immunology, supra).For example, the NZBxNZW F₁ mouse is an excellent model of human SLE,female mice develop high levels of anti-double- and single-stranded DNAautoantibodies, other anti-nuclear antibodies, and renal disease; deathusually occurs at approximately 8 months (see e.g., Theofilopoulos etal., Adv. Immunol. 37:269, 1985).

[0162] To evaluate the immunosuppressive effect of rHuAFP or a fragmentor analog thereof on SLE, i.e., the compound's ability of rHuAFP toprevent or ameliorate SLE, test compounds are administered to anappropriate animal, e.g., the NZBxNZW F₁ mouse, according to standardmethods, e.g., intravenously or intraperitoneally, at an appropriatedosage on a daily basis. Generally, administration is initiated prior tothe onset of SLE and/or after the clinical appearance of SLE. Controlanimals receive a placebo, e.g., human serum albumin, similarlyadministered as for rHuAFP or related molecules. The effect of the testcompound on SLE is monitored according to standard methods. For example,analysis of autoantibodies, e.g., anti-DNA antibodies can be monitored.If desired, histological inspection (e.g., by using any standardhistochemical or immunohistochemical procedure, see e.g., Ausubel etal., supra; Bancroft and Stevens, supra) of kidney tissue is performedand tissue samples examined microscopically for evidence of SLE, e.g.,evidence of lupus nephritis. Comparative studies between treated andcontrol animals are used to determine the relative efficacy of the testcompounds in preventing or ameliorating SLE. A molecule which preventsor ameliorates (decreases or suppresses or relieves or promotesremission of) the symptoms of SLE is considered useful in the invention.

[0163] Therapeutic Administration

[0164] As demonstrated above, recombinant alpha-fetoprotein, e.g.,rHuAFP (or a fragment or analog thereof) is effective in inhibitingproliferation of autoimmune cells and accordingly is useful for theprevention or amelioration of autoimmune diseases including, but notlimited to, multiple sclerosis, rheumatoid arthritis, diabetes mellitus,systemic lupus erythematosus, and myasthenia gravis. Accordingly,recombinant human alpha-fetoprotein (or a fragment or analog thereof)can be formulated according to known methods to prepare pharmaceuticallyuseful compositions.

[0165] Recombinant alpha-fetoprotein, e.g., rHuAFP (or a fragment oranalog thereof), is preferably administered to the patient in an amountwhich is effective in preventing or ameliorating the symptoms of anautoimmune disease. Generally, a dosage of 0.1 ng/kg to 10 g/kg body isadequate. If desired, administration is performed on a daily basis.Because there are no known adverse side effects related to recombinanthuman alpha-fetoprotein, it is believed that relatively high dosages canbe safely administered. For example, treatment of human patients will becarried out using a therapeutically effective amount of rHuAFP (or afragment or analog thereof) in a physiologically acceptable carrier.Suitable carriers and their formulation are described for example inRemington's Pharmaceutical Sciences by E. W. Martin. The amount ofrHuAFP to be administered will vary depending upon the manner ofadministration, the age and body weight of the patient, and with thetype of disease, and size of the patient predisposed to or sufferingfrom the disease. Preferable routes of administration include, forexample, subcutaneous, intravenous, intramuscular, or intradermalinjections which provide continuous, sustained levels of the drug in thepatient. In other preferred routes of administration, rHuAFP can begiven to a patient by injection or implantation of a slow releasepreparation, for example, in a slowly dissociating polymeric orcrystalline form; this sort of sustained administration can follow aninitial delivery of the drug by more conventional routes (for example,those described above). Alternatively, rHuAFP can be administered usingan infusion pump (e.g., an external or implantable infusion pump), thusallowing a precise degree of control over the rate of drug release, orthrough installation of rHuAFP in the nasal passages in a similarfashion to that used to promote absorption of insulin. As an alternativeto nasal transmucosal absorption, rHuAFP can be delivered by aerosoldeposition of the powder or solution into the lungs.

[0166] Furthermore, the method(s) of the invention can also employcombination therapy in which rHuAFP is administered eithersimultaneously or sequentially with a therapeutic agent such as ageneral or specific tolerizing agent (e.g., an anti-idiotypic agent(e.g., a monoclonal) or a therapeutic vaccine or an oral agent (e.g.,insulin, collagen or myelin basic protein) or a cytokine (e.g., Il-15)or an interferon (α-interferon) or an immunosuppressive agent.Preferably, an immunosuppressive agent is administered in an effectivedose which is lower than the standard dose when the immunosuppressiveagent is used by itself. Preferred immunosuppressive agents arecyclosporine, FK-506, steroids, azathioprine, or 15-deoxyspergualin.

[0167] Treatment is started generally with the diagnosis or suspicion ofan autoimmune disease and is generally repeated on a daily basis.Protection or prevention from the development (or progression orexacerbation) of an autoimmune disease is also achieved byadministration of rHuAFP prior to the onset of the disease. If desired,the efficacy of the treatment or protection regimens is assessed withthe methods of monitoring or diagnosing patients for autoimmune disease.

[0168] The method(s) of the invention can also be used to treatnon-human mammals, for example, domestic pets, or livestock.

Other Embodiments

[0169] In other embodiments, the invention includes the use of rHuAFP(or fragment or analog thereof) for the prevention or treatment ofacquired immunodeficiency syndrome (AIDS). To evaluate theimmunosuppressive effect of rHuAFP or a fragment or analog thereof onAIDS, i.e., the compound's ability to prevent or ameliorate anautoimmune component of AIDS, test compounds are administered to anappropriate animal (e.g., a human patient), according to standardmethods, e.g., intravenously or intraperitoneally, at an appropriatedosage on a daily basis as is discussed above. Generally, administrationis initiated prior to the onset of AIDS and/or after the clinicalappearance of AIDS. Control animals receive a placebo, e.g., human serumalbumin, similarly administered as for rHuAFP or related molecules. Theeffect of the test compound on AIDS is monitored according to standardmethods. For example, analysis of the ability of the test compound toinhibit or prevent or ameliorate the destruction of helper T cells canbe monitored. Comparative studies between treated and control animalsare used to determine the relative efficacy of the test compounds inpreventing or ameliorating AIDS. A molecule which prevents orameliorates (decreases or suppresses or relieves or promotes remissionof) the symptoms of AIDS is considered useful in the invention.

[0170] In the invention also includes the use of a therapeuticallyeffective amount rHuAFP (or fragment or analog thereof) for inhibitingthe rejection of a transplanted organ (e.g., the heart, the liver, thelung, the pancreas, and the kidney), tissue (e.g., skin, bone marrow,dura mater, bone, implanted collagen, an implanted bioreactor), or cell(e.g., β islet cells of the pancreas, stem cells, hematopoietic cells,lymph cells, neuroendocrine or adrenal cells) in a mammal. Suchtransplanted organs, tissues, or cells may be derived from any source,e.g., such biological material can be allogenic, phenogenic, autologous,synthetic, artificial or genetically-engineered. For example, the methodcan also be used when the patient is the recipient of an allograft sucha heart or kidney from another species.

[0171] In one working example, the immunosuppressive effect of rHuAFP onclinical transplantation, i.e., the ability of rHuAFP to prevent orameliorate transplant rejection (e.g., hyperacute rejection, acuterejection and chronic rejection), is evaluated by administering rHuAFPto an NIH minipig according to standard methods, e.g., intravenously orintraperitoneally, at an appropriate dosage on a daily basis. Generally,administration of rHuAFP is initiated prior to the transplant, e.g.,transplantation of a kidney and/or after the transplant procedure.Control animals receive a placebo, e.g., human serum albumin, similarlyadministered as for rHuAFP. The effect of rHuAFP on transplant rejectionis monitored according to standard methods. One manifestation of therejection process is diminished function of the transplanted organ, forexample, analysis of urine output can be monitored. If desired,histological inspection (e.g., by using any standard histochemical orimmunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroftand Stevens, supra) of kidney tissue is performed and tissue samplesobtained by biopsy are examined microscopically for evidence oftransplant rejection, e.g., chronic interstitial fibrosis, vascularthrombosis, or the presence of abnormal lymphocytic infiltrates.Comparative studies between treated and control animals are used todetermine the relative efficacy of rHuAFP in preventing or amelioratingtransplant rejection. Recombinant HuAFP (a fragment or analog thereof)which prevents or ameliorates (decreases or suppresses or relieves orpromotes remission of) the symptoms of transplant rejection isconsidered useful in the invention.

[0172] Graft-Versus-Host Disease

[0173] Studies of neonatal mice grafted with allogeneic T cellsindicates that treatment with rHuAFP has an inhibitory effect on GVHD.SCID mice at 3 days of age were used as recipients because of their lackof functional B and T cells. In this experimental system the grafted Tcells can react against the host whereas the recipient mice cannot mountan effective response against the graft. A SCID mouse (3 dayspost-natal) was injected i.p. with 100 μl PBS and 4 hours later with5×10⁶ spleen T cells from C57Bl/6 in 100 μl PBS on day 0. This mouse wasthereafter injected with 100 μl PBS day 1 and 3. The results from thismouse are presented in the B6 column. A SCID mouse (3 days post-natal)was injected i.p. with 200 μg rHuAFP in 100 μl PBP (100 μg rHuAFP pergram body weight) and 4 hours later with 5×10⁶ spleen T cells fromC56Bl/6 in 100 μl PBS on day 0. This mouse was thereafter injected with50 μg rHuAFP per gram body weight in 100 μl PBS on day 1 and 3. Theresults from this mouse are presented in the B6+AFP column.

[0174] In this experimental system loss of weight and decreased cellnumbers in the lymphoid organs are cardinal signs of severe GVHD.Differences could be detected in weight, as the mouse inj. B6+AFP gainedmore weight than the one inj. B6 (Table 2). The weight of the AFPtreated mouse was close to that of age-matched untreated SCID mice atour animal facilities. TABLE 2 Weight (g) inj inj Day B6 B6 + AFP 0 1.61.9 3 2.8 3.2 5 3.2 4.1

[0175] Moreover, at 5 days p.i. the total cell numbers in spleen, bonemarrow, and liver were lower in the mouse inj. B6 when compared to themouse inj. B6+AFP (Table 3). A SCID mouse received an allogenic T cellgraft in the absence (inj. B6) or presence (inj. B6+AFP) of rHuAFP asdescribed for FIG. 5. At 5 days post-injection, organ size wasdetermined an presented by the number cells in each tissue (cells×10⁻⁶).TABLE 3 Organ sizes (cells × 10⁻⁶) Organ inj. B6 inj. B6 + AFP Spleen 1232 Bone Marrow 6.0 9.2 Liver 0.56 1.0

[0176] At day 5 p.i. the mouse injected with C57Bl/6 only had a higherpercentage of exogenous T cells in spleen, bone marrow, and liver thanthe mouse treated with AFP as determined in FACS. Also, at thistime-point the amount of cells expressing CD19 and CD117 (c-kit) waslower in the liver of the mouse that was not treated with AFP both whencounted in percentage and total cell numbers which indicates a moresevere GVHD. When sera from the mice were analyzed for interferon-γcontent, an interleukin that is elevated during acute GVHD, theconcentration in mice inj. B6 (6.5 ng/ml) was twice as high as for miceinj. B6+AFP (3.1 ng/ml). Thus, these data from GVHD studies indicates aninhibitory effect of AFP on the allogeneic T cells and suppress GVHD.

[0177] Transplant rejection

[0178] Results

[0179] Enhanced MHC expression on BM cells cultured in the presence ofrHuAFP.

[0180] BM cells from adult C.B-17 mice cultured for three days in thepresence of optimal doses of IL-3 (2000 U/ml), IL-7 (1%), or rHuAFP (100μg/ml) were analyzed for expression of MHC class I (H-2K^(d)) and MHCclass II (I-A^(d)). Bone marrow cells (2.5×10⁶/ml) from C.B.-17 micewere cultured in 2 ml of f-DMEM medium containing 1% FCS in the presenceor absence of IL-3, IL-7, or rHuAFP. At 3 days of culture, cells wereharvested and stained with anti-H-2K^(d) and analyzed in FACS. Thefigure shows percent of cells with high expression of H-2K^(d) in mediumcontrol, IL-3, IL-7, and rHuAFP cultures in a representative experiment.As demonstrated in a representative experiment, a substantial increasein the intensity of MHC class I (MHC I) expression was seen on BM cellscultured in the presence of rHuAFP where 90% of the cells were MHCI^(high) compared to 61%, 40% and 33% for cells cultured with IL-3, IL-7and with medium only respectively (FIGS. 10A and 10B). In the individualexperiments, rHuAFP induced high intensity of MHC I expression onbetween 88% and 98% of the cells and interestingly, similar percentageswere seen even after a 10-fold decrease in the concentration of rHuAFP,i.e. to 10 μg/ml.

[0181] Despite the fact that the total cell numbers were not higher incultures supplemented with AFP than with IL-3 or IL-7 (FIG. 13), alsothe absolute numbers of cells with high expression of MHC I weresignificantly higher in the presence of AFP (FIG. 2B). (Schneider). Bonemarrow cells (10⁶ to 2.5×10⁶/ml) from C.B-17 mice were cultured in 2 mlof f-DMEM medium containing 1% FCS in the presence or absence of IL-3,IL-7, or rHuAFP. At 3 days of culture, cells were harvested and cellviability was determined with the trypan blue dye exclusion test. Thefigure shows mean percent standard deviation of viable cells at 3 daysof culture out of total number of seeded cells at day 0 as counted from4 separate experiments where the total number of seeded cells variedbetween 2×10⁶ to 5×10⁶ per well. The cells were cultured and treated asdescribed in FIG. 1A. The figure shows absolute numbers of cells withhigh expression of H-2K^(d) in medium control, IL-3, IL-7, and rHuAFPcultures in a representative experiment.

[0182] An increased frequency of MHC class II (MHC II) positive cellswas also seen in the presence of rHuAFP. Bone marrow cells from C.B-17mice were cultured as described in FIGS. 11A and 11B. At 3 days ofculture, cells were harvested and stained with anti-I-A^(d) and analyzedin FACS. The figure shows percent of cells positive for I-A^(d) inmedium control, IL-3, IL-7, and rHuAFP cultures in representativeexperiment. Flow cytometry analysis showed that 55% of the BM cells wereMHC II⁺ after three days of culture with rHuAFP (FIG. 11B) with wasnearly twice the percentage at day 0 (data not shown) and more than inthe medium control and in the presence of IL-3 and IL-7.

[0183] The absolute numbers of MHC II positive cells were similar inIL-7 and AFP cultures but significantly higher than in the mediumcontrol and IL-3 cultures (FIG. 11B). (Schneider) Bone marrow cells fromC.B-17 mice were cultured as described in FIG. 1A and harvested andanalyzed as described in FIG. 11A. The figures shows absolute numbers ofcells positive for I-A in medium control, IL-3, IL-7, and rHuAFPcultures in representative experiment.

[0184] There are numerous studies which have shown that AFP can exertgrowth regulatory effects of MHC class II expressing cells such asmonocytes and thyroid epithelia cells (Wang, et al. Hepatology22:921-928, 1995). We wanted to ascertain whether I-A^(k) expressingcells within whole bone marrow would be modulated upon co-culturing with100 μg/ml rHuAFP. Simultaneously, we investigated the effects of rHuAFPon MHC class I expressing cells within the adult BM. This was performedemploying fluorescein-conjugated anti-H-2K^(k) antibodies. Asillustrated in FIG. 12A, only a minority of cells in normal BM exhibit ahigh expression level of MHC molecules. When BM cells were cultured inthe presence of rHuAFP, there is a distinct pattern of stainingintensity. AFP increased the proportion of I-A^(k) expressing cells to40% versus 12% in control cultures of media alone or containingequivalent amounts of either mouse or human albumin. Cells defined asH-2K^(k, high) represented approximately 80% of the total BM analyzed incultures containing rHuAFP, illustrating a 15 fold increase over controlcultures with mouse and human albumin additions or no protein additions.

[0185] The effects of AFP on BM cell cultures cannot be reproduced withhuman or mouse albumin.

[0186] AFP share many physio-chemical properties with albumin, such asthe overall structures, including the three-domain structure, andcomparable binding properties. Therefore we determined whether theeffects in BM cells observed for AFP could also be ascribed to mouse orhuman albumin. For this purpose BM cells from CBA/J mice were culturedin the presence of 100 μg/ml of rHuAFP, human albumin, or mouse albumin.As demonstrated in FIG. 12B neither human or mouse albumin shared theproperties of AFP considering increase in MHC I or II intensity orenhanced frequencies of DN T cells and IgM positive B lineage cells.Instead, human and mouse albumin cultures were comparable to the mediumcontrol in these experiments.

EXPERIMENTAL

[0187] MATERIALS AND METHODS

[0188] Mice. C.B-17 (H-2^(d)) and CBA/J (H-2^(k)) mouse strains wereobtained from Bomholtsgaard, Denmark and wee then bred and maintained inour own animal facilities.

[0189] Preparation of hone marrow cells. Femurs and tibias were removedaseptically from mice and flushed with PBS using a syringe. Single cellsuspensions were then washed three times in PBS. Cell viability wasdetermined by the trypan blue dye exclusion test.

[0190] Membrane labeling of bone marrow cells with PKH67-G1. Bone marrowcells were labeled using the PKH67 Green Fluorescent Cell Linker Kit(PKH67-GL, Sigma Biosciences, St. Louis, Mo., U.S.A.). Briefly, cellswere diluted in Diluent C (2×10⁷ cells/ml) and then mixed with an equalvolume of 2×10⁻⁶ M PKH67 dye in Diluent C to a final concentration of10⁻⁶ M dye and 10⁷ cells/ml. After 2 minutes incubation at 25° C. thereaction was stopped by adding an equal volume of complete f-DMEM mediumcontaining 10% FCS. Cells were washed and analyzed on a FACScan® flowcytometer (Becton Dickinson, San Jose, CA) to determine labelingintensity.

[0191] In Vitro Cultures and Cell Proliferation. BM cells were culturedin 37° C. in a humidified atmosphere of 7.5% CO₂ in an incubator(Biocenter 2001, Salvis AG, Reussbuhl, Switzerland) in flat-bottomed 24well plates (A/S Nunc, Roskilde, Denmark) or round-bottomed 96 wellplates (Coming Costar, Acton, Mass., U.S.A.) in f-DMEM mediumsupplemented with 2 mM L-glutamine, 5×10⁻⁵ M 2-mercaptoethanol, and 10μg/ml gentamicin. The cultures were complemented with recombinant IL-3(Karasuyama & Melchers, Eur. J. Immunol. 18:97-104, 1998; kindlyprovided by Prof. Jan Andersson, Basel Institute for Immunology,Switzerland), crude supernatant from the IL-7 producing hybridomaJM-IL-7 (kindly provided by Dr. Jan Andersson), recombinant humanalpha-fetoprotein (Boismenu, et al. Adv. Exp. Med. Biol. 383:255-269,1995), human albumin, or mouse albumin and supplemented with 5 μg/mltransferrin, 1% fetal calf serum (FCS), or 0.5% autologous normal mouseserum (NMS). Cells from 24-well plates were harvested for flow cytometryanalysis and determined for cell viability by the trypan blue dyeexclusion test. Cell proliferation was determined by incubatingtriplicate cultures in round-bottomed 96 well plates with 1 μCi/ cultureof ³H-thymidine (Amersham International plc, Amersham, UK; spec. act.,925 Gbq/mmol) for 4 h prior to harvest onto glassfiber filters in amultiple cell harvester (1295-004 Betaplate® Pharmacia LKB, Uppsala,Sweden). Radioactivity on dried filters was measured by scintillationcounting in a beta counter (1205 Betaplate®, Pharmacia LKB, Uppsala,Sweden).

[0192] Flow cytometry analysis. If not otherwise stated, the mAbs wereobtained from PharMingen, San Diego, Calif.. All steps were carried outat 4° C. Cells (10⁵ to 10⁶ per sample) were pre-incubated for 30 minuteswith 50 μl crude supernatant from 2.4 G2 hybridomas, washed once in 250μl PBS and then stained for 30 minutes with pretitered concentrations ofthe following mABs (obtained from Pharmigen) in 50 μl of PBS:FITC-labeled anti-H-2K^(d) (SF1- 1.1), biotinylated anti-I-A^(d)(AMS-32.1), FITC-labeled anti-H-2K^(k) (AF2-12.1), and biotinylatedanti-I-A^(k) (11-5.2). The cells were then washed once in 250 μ PBS andcells stained with biotinylated mAb were incubated for 25 min werestreptavidin-PE (Becton Dickinson, San Jose, Calif.) followed by threewashes with 250 μl of PBS. The samples were diluted to a final volume of0.5 ml in PBS containing 1 μg/ml of propidium iodide and analyzed on aFACScan® flow cytometer (Becton Dickinson, San Jose, Calif.). An amountof 5-20×10³ cells were collected per sample using a FSC vs. SSC livegate to ignore erthrocytes and an FL3 vs. FL2 live gate to exclude deadpropidium iodide stained cells.

[0193] Treatment of BM cells with albumin. Bone marrow cells (2×10⁶/ml)from CBA/J mice were cultured in 2 ml of f-DMEM medium containing 1% FCSin the presence or absence of rHuAFP, human albumin (HuAlb), or mousealbumin (MoAlb). At 4 days of culture, cells were harvested, stainedwith anti-H-2K^(k), and analyzed in FACS. FIG. 12B shows percent cellsthat were H-2K^(k,high) in medium without added AFP or supplemented witheither rHuAFP, HuAlb, or MoAlb. FIG. 12B shows percent cells that were1-A^(k+) in medium without added AFP or supplemented with rHuAFP, HuAlb,or MoAlb.

[0194] Bone marrow reconstitution in the presence of AFP in vitro and invivo

[0195] Enhanced BM cell recovery in the presence of rHuAFP.

[0196] The impact of AFP to enhance BM cell reconstitution followingsublethal gamma irradiation was analyzed by irradiating BM cells fromC.B-17 mice with a dose of 600 rad. Thereafter, irradiated andnon-irradiated cells were cultured in the absence or presence of IL-7 orrHuAFP. After 3 days of culture cells were harvested and cell densitieswere determined by the trypan blue dye exclusion test. Consistent withthe data presented in FIG. 13 the highest cell density fornon-irradiated cells was observed in the IL-7 cultures (FIG. 14A). BMcells from C.B- 17 mice were irradiated with 600 rad. Thereafter, theirradiated and non-irradiated cells were cultured in the absence orpresence of IL-7 or rHuAFP. After 3 days of culture, cells wereharvested and cell densities were determined by the trypan blue dayexclusion test. Conversely, following irradiation there was asignificant enhancement in cell recovery among cells cultured in thepresence of 100 μg/ml of rHuAFP (FIG. 14B).

[0197] All publications, manufacturer's instructions, patents, andpatent applications mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1 16 1 2027 DNA Homo sapiens 1 atattgtgct tccaccactg ccaataacaaaataactagc aaccatgaag tgggtggaat 60 caattttttt aattttccta ctaaattttactgaatccag aacactgcat agaaatgaat 120 atggaatagc ttccatattg gattcttaccaatgtactgc agagataagt ttagctgacc 180 tggctaccat attttttgcc cagtttgttcaagaagccac ttacaaggaa gtaagcaaaa 240 tggtgaaaga tgcattgact gcaattgagaaacccactgg agatgaacag tcttcagggt 300 gtttagaaaa ccagctacct gcctttctggaagaactttg ccatgagaaa gaaattttgg 360 agaagtacgg acattcagac tgctgcagccaaagtgaaga gggaagacat aactgttttc 420 ttgcacacaa aaagcccact gcagcatggatcccactttt ccaagttcca gaacctgtca 480 caagctgtga agcatatgaa gaagacagggagacattcat gaacaaattc atttatgaga 540 tagcaagaag gcatcccttc ctgtatgcacctacaattct tctttcggct gctgggtatg 600 agaaaataat tccatcttgc tgcaaagctgaaaatgcagt tgaatgcttc caaacaaagg 660 cagcaacagt tacaaaagaa ttaagagaaagcagcttgtt aaatcaacat gcatgtccag 720 taatgaaaaa ttttgggacc cgaactttccaagccataac tgttactaaa ctgagtcaga 780 agtttaccaa agttaatttt actgaaatccagaaactagt cctggatgtg gcccatgtac 840 atgagcactg ttgcagagca gatgtgctggattgtctgca ggatggggaa aaaatcatgt 900 cctacatatg ttctcaacaa gacactctgtcaaacaaaat aacagaatgc tgcaaactga 960 ccacgctgga acgtggtcaa tgtataattcatgcagaaaa tgatgaaaaa cctgaaggtc 1020 tatctccaaa tctaaacagg tttttaggagatagagattt taaccaattt tcttcagggg 1080 aaaaaaatat cttcttggca agttttgttcatgaatattc aagaagacat cctcagcttg 1140 ctgtctcagt aattctaaga gttgctaaaggataccagga gttattggag aagtgtttcc 1200 agactgaaaa ccctcttgaa tgccaagataaaggagaaga agaattacag aaatacatcc 1260 aggagagcca agcattggca aagcgaagctgcggcctctt ccagaaacta ggagaatatt 1320 acttacaaaa tgagtttctc gttgcttacacaaagaaagc cccccagctg acctcgtcgg 1380 agctgatggc catcaccaga aaaatggcagccacagcagc cacttgttgc caactcagtg 1440 aggacaaact attggcctgt ggcgagggagcggctgacat tattatcgga cacttatgta 1500 tcagacatga aatgactcca gtaaaccctggtgttggcca gtgctgcact tcttcatatg 1560 ccaacaggag gccatgcttc agcagcttggtggtggatga aacatatgtc cctcctgcat 1620 tctctgatga caagttcatt ttccataaggatctgtgcca agctcagggt gtagcgctgc 1680 aaaggatgaa gcaagagttt ctcattaaccttgtgaagca aaagccacaa ataacagagg 1740 aacaacttga ggctctcatt gcagatttctcaggcctgtt ggagaaatgc tgccaaggcc 1800 aggaacagga agtctgcttt gctgaagagggacaaaaact gatttcaaaa actggtgctg 1860 ctttgggagt ttaaattact tcaggggaagagaagacaaa acgagtcttt cattcggtgt 1920 gaacttttct ctttaatttt aactgatttaacactttttg tgaattaatg aaatgataaa 1980 gacttttatg tgagatttcc ttatcacagaaataaaatat ctccaaa 2027 2 590 PRT Homo sapiens 2 Thr Leu His Arg Asn GluTyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr 1 5 10 15 Gln Cys Thr Ala GluIle Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe 20 25 30 Ala Gln Phe Val GlnGlu Ala Thr Tyr Lys Glu Val Ser Lys Met Val 35 40 45 Lys Asp Ala Leu ThrAla Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser 50 55 60 Ser Gly Cys Leu GluAsn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys 65 70 75 80 His Glu Lys GluIle Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser 85 90 95 Gln Ser Glu GluGly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro 100 105 110 Thr Ala AlaTrp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser 115 120 125 Cys GluAla Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile 130 135 140 TyrGlu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu 145 150 155160 Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala 165170 175 Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys180 185 190 Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro ValMet 195 200 205 Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val ThrLys Leu 210 215 220 Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile GlnLys Leu Val 225 230 235 240 Leu Asp Val Ala His Val His Glu His Cys CysArg Ala Asp Val Leu 245 250 255 Asp Cys Leu Gln Asp Gly Glu Lys Ile MetSer Tyr Ile Cys Ser Gln 260 265 270 Gln Asp Thr Leu Ser Asn Lys Ile ThrGlu Cys Cys Lys Leu Thr Thr 275 280 285 Leu Glu Arg Gly Gln Cys Ile IleHis Ala Glu Asn Asp Glu Lys Pro 290 295 300 Glu Gly Leu Ser Pro Asn LeuAsn Arg Phe Leu Gly Asp Arg Asp Phe 305 310 315 320 Asn Gln Phe Ser SerGly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val 325 330 335 His Glu Tyr SerArg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu 340 345 350 Arg Val AlaLys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr 355 360 365 Glu AsnPro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys 370 375 380 TyrIle Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe 385 390 395400 Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr 405410 415 Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr420 425 430 Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser GluAsp 435 440 445 Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile IleGly His 450 455 460 Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro GlyVal Gly Gln 465 470 475 480 Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg ProCys Phe Ser Ser Leu 485 490 495 Val Val Asp Glu Thr Tyr Val Pro Pro AlaPhe Ser Asp Asp Lys Phe 500 505 510 Ile Phe His Lys Asp Leu Cys Gln AlaGln Gly Val Ala Leu Gln Arg 515 520 525 Met Lys Gln Glu Phe Leu Ile AsnLeu Val Lys Gln Lys Pro Gln Ile 530 535 540 Thr Glu Glu Gln Leu Glu AlaLeu Ile Ala Asp Phe Ser Gly Leu Leu 545 550 555 560 Glu Lys Cys Cys GlnGly Gln Glu Gln Glu Val Cys Phe Ala Glu Glu 565 570 575 Gly Gln Lys LeuIle Ser Lys Thr Gly Ala Ala Leu Gly Val 580 585 590 3 197 PRT Homosapiens 3 Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp SerTyr 1 5 10 15 Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr IlePhe Phe 20 25 30 Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser LysMet Val 35 40 45 Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp GluGln Ser 50 55 60 Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu GluLeu Cys 65 70 75 80 His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser AspCys Cys Ser 85 90 95 Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala HisLys Lys Pro 100 105 110 Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pro GluPro Val Thr Ser 115 120 125 Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr PheMet Asn Lys Phe Ile 130 135 140 Tyr Glu Ile Ala Arg Arg His Pro Phe LeuTyr Ala Pro Thr Ile Leu 145 150 155 160 Leu Ser Ala Ala Gly Tyr Glu LysIle Ile Pro Ser Cys Cys Lys Ala 165 170 175 Glu Asn Ala Val Glu Cys PheGln Thr Lys Ala Ala Thr Val Thr Lys 180 185 190 Glu Leu Arg Glu Ser 1954 192 PRT Homo sapiens 4 Ser Leu Leu Asn Gln His Ala Cys Pro Val Met LysAsn Phe Gly Thr 1 5 10 15 Arg Thr Phe Gln Ala Ile Thr Val Thr Lys LeuSer Gln Lys Phe Thr 20 25 30 Lys Val Asn Phe Thr Glu Ile Gln Lys Leu ValLeu Asp Val Ala His 35 40 45 Val His Glu His Cys Cys Arg Ala Asp Val LeuAsp Cys Leu Gln Asp 50 55 60 Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser GlnGln Asp Thr Leu Ser 65 70 75 80 Asn Lys Ile Thr Glu Cys Cys Lys Leu ThrThr Leu Glu Arg Gly Gln 85 90 95 Cys Ile Ile His Ala Glu Asn Asp Glu LysPro Glu Gly Leu Ser Pro 100 105 110 Asn Leu Asn Arg Phe Leu Gly Asp ArgAsp Phe Asn Gln Phe Ser Ser 115 120 125 Gly Glu Lys Asn Ile Phe Leu AlaSer Phe Val His Glu Tyr Ser Arg 130 135 140 Arg His Pro Gln Leu Ala ValSer Val Ile Leu Arg Val Ala Lys Gly 145 150 155 160 Tyr Gln Glu Leu LeuGlu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu 165 170 175 Cys Gln Asp LysGly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser 180 185 190 5 201 PRTHomo sapiens 5 Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys LeuGly Glu 1 5 10 15 Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr LysLys Ala Pro 20 25 30 Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg LysMet Ala Ala 35 40 45 Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys LeuLeu Ala Cys 50 55 60 Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His Leu CysIle Arg His 65 70 75 80 Glu Met Thr Pro Val Asn Pro Gly Val Gly Gln CysCys Thr Ser Ser 85 90 95 Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Leu ValVal Asp Glu Thr 100 105 110 Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys PheIle Phe His Lys Asp 115 120 125 Leu Cys Gln Ala Gln Gly Val Ala Leu GlnArg Met Lys Gln Glu Phe 130 135 140 Leu Ile Asn Leu Val Lys Gln Lys ProGln Ile Thr Glu Glu Gln Leu 145 150 155 160 Glu Ala Leu Ile Ala Asp PheSer Gly Leu Leu Glu Lys Cys Cys Gln 165 170 175 Gly Gln Glu Gln Glu ValCys Phe Ala Glu Glu Gly Gln Lys Leu Ile 180 185 190 Ser Lys Thr Gly AlaAla Leu Gly Val 195 200 6 389 PRT Homo sapiens 6 Thr Leu His Arg Asn GluTyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr 1 5 10 15 Gln Cys Thr Ala GluIle Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe 20 25 30 Ala Gln Phe Val GlnGlu Ala Thr Tyr Lys Glu Val Ser Lys Met Val 35 40 45 Lys Asp Ala Leu ThrAla Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser 50 55 60 Ser Gly Cys Leu GluAsn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys 65 70 75 80 His Glu Lys GluIle Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser 85 90 95 Gln Ser Glu GluGly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro 100 105 110 Thr Ala AlaTrp Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser 115 120 125 Cys GluAla Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile 130 135 140 TyrGlu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu 145 150 155160 Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pro Ser Cys Cys Lys Ala 165170 175 Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys180 185 190 Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Pro ValMet 195 200 205 Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val ThrLys Leu 210 215 220 Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile GlnLys Leu Val 225 230 235 240 Leu Asp Val Ala His Val His Glu His Cys CysArg Ala Asp Val Leu 245 250 255 Asp Cys Leu Gln Asp Gly Glu Lys Ile MetSer Tyr Ile Cys Ser Gln 260 265 270 Gln Asp Thr Leu Ser Asn Lys Ile ThrGlu Cys Cys Lys Leu Thr Thr 275 280 285 Leu Glu Arg Gly Gln Cys Ile IleHis Ala Glu Asn Asp Glu Lys Pro 290 295 300 Glu Gly Leu Ser Pro Asn LeuAsn Arg Phe Leu Gly Asp Arg Asp Phe 305 310 315 320 Asn Gln Phe Ser SerGly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val 325 330 335 His Glu Tyr SerArg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu 340 345 350 Arg Val AlaLys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr 355 360 365 Glu AsnPro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys 370 375 380 TyrIle Gln Glu Ser 385 7 393 PRT Homo sapiens 7 Ser Leu Leu Asn Gln His AlaCys Pro Val Met Lys Asn Phe Gly Thr 1 5 10 15 Arg Thr Phe Gln Ala IleThr Val Thr Lys Leu Ser Gln Lys Phe Thr 20 25 30 Lys Val Asn Phe Thr GluIle Gln Lys Leu Val Leu Asp Val Ala His 35 40 45 Val His Glu His Cys CysArg Ala Asp Val Leu Asp Cys Leu Gln Asp 50 55 60 Gly Glu Lys Ile Met SerTyr Ile Cys Ser Gln Gln Asp Thr Leu Ser 65 70 75 80 Asn Lys Ile Thr GluCys Cys Lys Leu Thr Thr Leu Glu Arg Gly Gln 85 90 95 Cys Ile Ile His AlaGlu Asn Asp Glu Lys Pro Glu Gly Leu Ser Pro 100 105 110 Asn Leu Asn ArgPhe Leu Gly Asp Arg Asp Phe Asn Gln Phe Ser Ser 115 120 125 Gly Glu LysAsn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg 130 135 140 Arg HisPro Gln Leu Ala Val Ser Val Ile Leu Arg Val Ala Lys Gly 145 150 155 160Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu 165 170175 Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser 180185 190 Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu195 200 205 Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys AlaPro 210 215 220 Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys MetAla Ala 225 230 235 240 Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp LysLeu Leu Ala Cys 245 250 255 Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly HisLeu Cys Ile Arg His 260 265 270 Glu Met Thr Pro Val Asn Pro Gly Val GlyGln Cys Cys Thr Ser Ser 275 280 285 Tyr Ala Asn Arg Arg Pro Cys Phe SerSer Leu Val Val Asp Glu Thr 290 295 300 Tyr Val Pro Pro Ala Phe Ser AspAsp Lys Phe Ile Phe His Lys Asp 305 310 315 320 Leu Cys Gln Ala Gln GlyVal Ala Leu Gln Arg Met Lys Gln Glu Phe 325 330 335 Leu Ile Asn Leu ValLys Gln Lys Pro Gln Ile Thr Glu Glu Gln Leu 340 345 350 Glu Ala Leu IleAla Asp Phe Ser Gly Leu Leu Glu Lys Cys Cys Gln 355 360 365 Gly Gln GluGln Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Leu Ile 370 375 380 Ser LysThr Gly Ala Ala Leu Gly Val 385 390 8 325 PRT Homo sapiens 8 Met Ser TyrIle Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr 1 5 10 15 Glu CysCys Lys Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His 20 25 30 Ala GluAsn Asp Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg 35 40 45 Phe LeuGly Asp Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn 50 55 60 Ile PheLeu Ala Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln 65 70 75 80 LeuAla Val Ser Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu 85 90 95 LeuGlu Lys Cys Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys 100 105 110Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala 115 120125 Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln 130135 140 Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser145 150 155 160 Ser Glu Leu Met Ala Ile Thr Arg Lys Met Ala Ala Thr AlaAla Thr 165 170 175 Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Ala Cys GlyGlu Gly Ala 180 185 190 Ala Asp Ile Ile Ile Gly His Leu Cys Ile Arg HisGlu Met Thr Pro 195 200 205 Val Asn Pro Gly Val Gly Gln Cys Cys Thr SerSer Tyr Ala Asn Arg 210 215 220 Arg Pro Cys Phe Ser Ser Leu Val Val AspGlu Thr Tyr Val Pro Pro 225 230 235 240 Ala Phe Ser Asp Asp Lys Phe IlePhe His Lys Asp Leu Cys Gln Ala 245 250 255 Gln Gly Val Ala Leu Gln ArgMet Lys Gln Glu Phe Leu Ile Asn Leu 260 265 270 Val Lys Gln Lys Pro GlnIle Thr Glu Glu Gln Leu Glu Ala Leu Ile 275 280 285 Ala Asp Phe Ser GlyLeu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln 290 295 300 Glu Val Cys PheAla Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Gly 305 310 315 320 Ala AlaLeu Gly Val 325 9 30 DNA Homo sapiens 9 aaaaaaggta ccacactgca tagaaatgaa30 10 33 DNA Homo sapiens 10 aaaaaaggat ccttagcttt ctcttaattc ttt 33 1133 DNA Homo sapiens 11 aaaaaaatcg atatgagctt gttaaatcaa cat 33 12 33 DNAHomo sapiens 12 aaaaaaggat ccttagctct cctggatgta ttt 33 13 33 DNA Homosapiens 13 aaaaaaatcg atatgcaagc attggcaaag cga 33 14 33 DNA Homosapiens 14 aaaaaaggat ccttaaactc ccaaagcagc acg 33 15 33 DNA Homosapiens 15 aaaaaaatcg atatgtccta catatgttct caa 33 16 9 PRT Homo sapiens16 Ser Tyr Ile Cys Ser Gln Gln Asp Thr 1 5

What is claimed is:
 1. A method of inhibiting transplant rejection in amammal, said method comprising administering to said mammal atherapeutically effective amount of recombinant human alpha-fetoproteinor a fragment thereof.
 2. A method of inhibiting graft-versus-hostdisease in a mammal, said method comprises administering to said mammala therapeutically effective amount of recombinant humanalpha-fetoprotein or a fragment thereof.
 3. A method of mitigating theside effects in a mammal undergoing chemotherapy, said method comprisingadministering to said mammal a therapeutically effective amount ofrecombinant human alpha-fetoprotein or a fragment thereof.
 4. A methodof mitigating the side effects in a mammal exposed to irradiationtherapy, said method comprising administering to said mammal atherapeutically effective amount of recombinant human alpha-fetoproteinor a fragment thereof.
 5. The method of claims 1, 2, 3, or 4, whereinthe said alpha-fetoprotein fragment comprising Domain I, Domain II,Domain III, Domain I+II, Domain II+III, or Fragment I.
 6. The method ofclaims 1, 2, 3, or 4, wherein said alpha-fetoprotein or fragment thereofis expressed in baculovirus.
 7. The method of claims 1, 2, 3, or 4,wherein said alpha-fetoprotein or fragment thereof is expressed in aeukaryote.
 8. The method of claims 1, 2, 3, or 4, wherein saidalpha-fetoprotein or fragment thereof is expressed as a transgene. 9.The method of claims 1, 2, 3, or 4, wherein said alpha-fetoprotein orfragment thereof is expressed in E. coli.
 10. The method of claims 1, 2,3, or 4, wherein the said alpha-fetoprotein or fragment thereof isunglycosylated.
 11. The method of claims 1, 2, 3, or 4, wherein the saidalpha-fetoprotein or fragment thereof is glycosylated.
 12. The method ofclaims 1, 2, 3, or 4, wherein said mammal is a human.